Vous êtes sur la page 1sur 11

Rheol Acta (2016) 55:485495

DOI 10.1007/s00397-015-0894-3


Blood linear viscoelasticity by small amplitude oscillatory flow

Giovanna Tomaiuolo 1,2 & Antonio Carciati 1,2 & Sergio Caserta 1,2 & Stefano Guido 1,2

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

Dipartimento di Ingegneria Chimica, dei Materiali e della Produzione

Industriale, Universit di Napoli Federico II, Naples, Italy

CEINGE Biotecnologie avanzate, Naples, Italy

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

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


(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

Rheol Acta (2016) 55:485495

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

Rheol Acta (2016) 55:485495

flow are sparse in the literature and currently limited. Thurston

(Thurston 1972; Thurston 1975) measured human blood by
using oscillatory tube flow, but blood viscoelastic properties
were not expressed in terms of G (storage modulus) and G
(loss modulus). In the same years, Chien (Chien et al. 1975)
applied oscillatory shear to human blood and RBC suspensions in a Weissenberg rheogoniometer with a cone and plate
geometry in a rather limited range of frequency [0.011 Hz].
The same experimental arrangement has been used by Drasler
et al. in a wider range of frequencies [0.007660 Hz] to investigate the viscoelastic behavior of concentrate suspensions of
RBCs (up to 95 % Hct) in order to allow close cell-cell contacts and to overcome the problem of cellular sedimentation
(Drasler et al. 1987). The results show a strong dependence of
G on frequency. Moreover, it was found that at the high Hct
values investigated, the suspending media (i.e., plasma, buffered saline and dextran solution) did not influence the mechanism of cell packing, suggesting that the latter could be affected by and/or related to the elastic modulus of RBC membrane. Measurements on RBCs hardened by glutaraldehyde
provided higher G values and a reduced dependence on frequency, showing that stiff cells can store more energy than
normal ones. More recently, blood viscoelastic behavior at
43 % Hct has been investigated by using cone-plate and parallel plate geometries (Alves et al. 2013). Moreover, passive
microrheology with the purpose to develop blood analogs
(Campo-Deao et al. 2013) and large amplitude oscillatory
shear (Sousa et al. 2013) have been used. From the modeling
point of view, starting from the Casson model and the Merril
and Pelletier steady state data (Merrill and Pelletier 1967;
Robertson et al. 2009), Apostolidis et al. (Apostolidis et al.
2015) introduced three additional parameters and found good
agreement with recent large amplitude oscillatory shear data
(Sousa et al. 2013). However, an extensive experimental study
of blood rheology in the linear viscoelastic regime by classical
oscillatory rheometry, including the effect of Hct and aggregating media, is still missing.
Here, we present the first systematic experimental investigation of human blood transient behavior in the linear viscoelastic regime by performing oscillatory shear measurements
through conventional bulk rheology. The storage (G) and loss
(G) moduli of whole normal human blood (Hct around 45 %)
have been measured over an extended range of frequencies
[0.130 rad/s]. Results shown that the loss modulus G predominates over the elastic modulus G across the entire range
of frequency explored, suggesting a behavior close to that of
weakly attractive suspensions. In order to elucidate the mechanism at work in oscillatory flow behavior of blood, the viscoelastic response has been measured at increasing RBC volume fractions and in suspension of RBCs in aggregating media (i.e., dextran solution in two different concentrations of
dextran). These findings could help in elucidating the significance of blood viscoelasticity alterations in pathological


situations, where increased hematocrit levels and aggregability are found.

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
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)


Rheol Acta (2016) 55:485495

Whole blood in physiological conditions (T = 37 C and Hct

about 45 %) was subjected to steady-state deformations at
controlled shear stress. The trend of blood viscosity of five
healthy donors is represented by the master curve shown in
Fig. 1a, where the average viscosity of 20 tests is plotted as a
function of shear rate and the error bars represent the standard
deviation. The well-known shear thinning behavior of whole
blood (black circles) is observed. The rheological behavior of
aggregating suspensions, such as whole blood, shows timedependent effects due to disruption and formation of RBC
aggregates under flow. For this reason, in the development
of the experimental protocol, blood samples have been subjected to pre-shear (200/s for 20 s) in order to promote the
homogeneity of the sample and to reduce any memory effects
(Snabre and Mills 1996). The effect of pre-shear on blood
flow curve is shown in Fig. 1a (open circles): at low shear
rates, with the initial pre-conditioning step, viscosity values
are higher than the ones in the sample without pre-shear,

indicating the presence of memory effects and the (partial)

breakage of the static microstructure made by RBC aggregates. At shear rates higher than about 5/s, the pre-shear effect
disappears, likely due to flow-induced disruption of RBC aggregates. Sedimentation and possible syneresis (leading to the
formation of a plasma layer near the surface) have been investigated by increasing and subsequently decreasing the imposed shear stress (black and gray triangles, respectively)
without reloading the sample and without pre-shear, as shown
in the inset of Fig. 1a. When the test is carried out from high to
low shear rates (up-down curve in the inset), the slowing
down of the flow promotes RBC aggregation. This results in
the onset of sedimentation at the lowest shear rates, which in
turn leads to a slight discrepancy between the down-up and
up-down curves, often referred to as the thixotropic behavior
of blood. To avoid this effect, only the increasing shear rate
flow behavior (down-up curve), without the pre-shearing step,
is considered as representative of blood viscosity.
Another peculiar feature of blood, indicating its nonNewtonian nature, is the presence of a yield stress, which is
also found in other concentrated suspensions (Baskurt 2007).
Whole blood yield stress has been confirmed experimentally
by direct measurements (Picart et al. 1998) and by using the
Casson model (Robertson et al. 2009). Experiments carried
out at low shear rates allow one to apply Casson equation to
determine the yield stress value. The data shown in Fig. 1a
have been rearranged in the so-called Casson plot (i.e., square
root of shear stress vs square root of shear rate), as shown in
Fig. 1b. Data points have been fitted by linear regression
(r2 > 0.99), and extrapolation to zero shear rate returned the
square root of the yield stress. Thus, the yield stress values for
measures with and without pre-shear have been estimated
equal to 5.2 and 3.8 mPa, respectively. These values are in
good agreement with the ones (ranging from 1.5 to 5 mPa)
reported in previous works in the literature, both experimental
(Chien et al. 1966) and theoretical (Fedosov et al. 2011).

Fig. 1 a Reference curves of whole blood viscosity as a function of shear

rate, by applying a pre-shear (open circle) and in absence of pre-shear
(dark circle). In the inset, black triangle represents the down-up curve,

gray triangle represents up-down curve. b Casson plot of the data

presented in a, without pre-shear. The continuous line is a linear fit of
the experimental data

ranging from 0.5 to 300/s. Viscosity curves were recorded by

using 30 measuring data points with sampling time of 10 s.
Oscillatory shear tests have been performed in order to
evaluate blood storage modulus G and loss modulus G.
Each oscillatory measurement has been preceded by strain
amplitude sweep test at 1 rad/s and 10 rad/s for strain amplitudes from 0.1 to 100 %, in order to determine the linear
viscoelastic regime. The frequency sweep tests were performed from 30 to 0.1 rad/s and strain amplitude from 7 %
to 10 %, depending on the data provided by amplitude test.
The number of data points per interval ranged from 8 to 16,
depending on the type of sample examined.

Whole blood steady-state behavior

Rheol Acta (2016) 55:485495


Fig. 2 a Storage modulus (black square) and loss modulus (black

triangle) as a function of angular frequency. The gray dashed lines
represent a power law fitting of the experimental data (R2 = 0.999), the

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 %

Whole blood linear viscoelasticity

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

Fig. 3 G and G as a function of time for four different angular


Fig. 4 Viscosity of RBC suspensions of different volume fraction as a

function of shear rate. In the inset, viscosity as a function of Hct at four
different shear rates is reported


Rheol Acta (2016) 55:485495

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

allowing optimal microcirculation and tissue perfusion. The

small magnitude of the error bars in Figs. 2 shows the high
reproducibility of our measurements, despite the difficulty in
handling a complex biological fluid such as blood. In fact, one
of the main issues to take into account is the role of RBC
sedimentation, which could be especially relevant in the small
oscillatory deformation regime (710 %) at low frequency. In
order to assess the impact of RBC sedimentation on our measurements, G and G have been measured as a function of
time, as reported in Fig. 3. It can be noticed that G and G
variations with time are rather limited (12.5 % for G, 6.7 %
for G), indicating that kinetic of RBC sedimentation is negligible for the duration of the test (about 13 min), as also
demonstrated in (Fabry 1987), where it is reported that sedimentation becomes significant only after 20 min in static conditions. Thus, the higher magnitude of error bars at the lower
Fig. 6 Comparison of viscosity
and magnitude of complex
viscosity by applying the CoxMerz rule for a Hct = 45 %, b
Hct = 60 %, c Hct = 75 %, and d
Hct = 90 %

frequencies could be explained by the proximity to the instrument detection limit due to the low viscosity of blood.

The effect of RBC volume fraction (Hct)

The evaluation of the rheological behavior of concentrated
RBC suspensions (i.e., Hct higher than the physiological value of 45 %) is an important pathophysiological issue. In some
blood disorders, such as polycythemia and myeloproliferative
neoplasm, RBC volume fraction is increased (Hct about
60 %), causing impaired blood flow at the microcirculation
level and thrombotic events (Adams et al. 2010; Kwaan and
Wang 2003; Sarkar and Rosenkrantz 2008). Moreover, in
blood bags used for RBC transfusion, Hct can reach values
higher than 90 %.

Rheol Acta (2016) 55:485495


Fig. 7 a The loss modulus G vs

the elastic modulus G and b the
loss tangent tan as a function of
angular frequency for three RBC
suspensions of different volume
fractions. The solid line represents
the line bisector

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
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.

One more viscoelastic parameter has been considered to

investigate the effect of increasing RBC volume fraction,
i.e., the loss tangent (tan ) as a function of angular frequency.
Tan , being the ratio of G to G, represents the ratio of the
viscous to the elastic response (energy loss/energy stored) of
the materials, and it can be used to quantify the presence and
extent of elasticity in a material. In Fig. 7b it is shown that tan
is higher than 1 for all the samples examined in the entire
range of frequency, but it decreases with RBC volume fraction. This trend confirms that the behavior of RBC suspensions approaches the one of an elastic solid when the RBC
volume fraction increases.

The effect of RBC aggregation

The tendency of RBCs to form packed structures plays an
important role in blood flow behavior, causing the increase
of blood viscosity, especially at low shear rates. It is well
known that the formation of 3D structures made by trains of
RBCs (rouleaux) is dependent on both the size of rouleaux
and the cohesive forces within aggregates, usually expressed
in terms of the shear stress required to disperse them.
Furthermore, RBC aggregation is promoted by the presence

Fig. 8 Viscosity of whole blood added with dextran at two

concentrations (1.5 and 3 mg/ml) as a function of shear rate. In the
inset, circles represent the down-up curve, the triangles represent the
up-down curve


Rheol Acta (2016) 55:485495

Fig. 9 a G and G as a function

of angular frequencies for whole
blood (black) and whole blood
with dextran at two
concentrations (1.5 mg/ml (gray)
and 3 mg/ml (white)). b Strain
amplitude sweep test at 10 rad/s

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

Rheol Acta (2016) 55:485495


Fig. 11 a The loss modulus G

vs the elastic modulus G and b
the loss tangent tan as a function
of angular frequency for whole
blood (black) and whole blood
added with dextran at two
concentrations (1.5 mg/ml (gray)
and 3 mg/ml (white)). The solid
line represents the line bisector

molecular weight species. Thus, the different curves in

Fig. 11a show a different sensitivity of the moduli on such
material parameters depending on dextran concentration (once
again, the solid line is a plot of G = G). In Fig. 10b, tan as a
function of angular frequency takes values higher than 1 for all
the samples examined in the entire range of frequency: tan
decreases with dextran concentration, confirming that the behavior of RBC suspensions approaches the one of elastic
solids when the adhesion strength, promoted by the presence
of dextran, increases.
Finally, a comparison between our data and results from the
literature is shown in Fig. 12, where black symbols refer to
this work, gray symbols to Alves et al. (Alves et al. 2013) and
open symbols to Campo-Deano et al. (Campo-Deao et al.
2013). The results of Alves et al. were obtained in a rotational
rheometer under oscillatory flow with an amplitude of 20 %
that, according to Fig. 2, is beyond the linear regime.
Notwithstanding, a reasonable agreement with our data is
found, at least in the range 110 rad/s. Concerning the work
of Campo-Deano et al., the good agreement found at low
frequencies (210 rad/s) confirms that blood microstructure
is essentially the same both in the static conditions encountered in microrheology and at low frequencies in bulk rheology. Thus, in both the experimental conditions, blood microstructure is essentially the same, and it likely consists of more
or less aggregated RBCs. One could speculate that in
microrheology experiments, RBC aggregates limit bead

Fig. 12 Comparison between experimental data at Hct = 45 % and

results from the literature

motion and this confining effect is the source of viscoelasticity

found by Campo-Deano et al. (Campo-Deao et al. 2013). On
the other hand, at higher values of frequency, red blood cell
aggregates are probably broken in bulk rheology, and blood
microstructure becomes significantly different from the one
present under static conditions. This could explain the discrepancy between our data and the results of Campo-Deano et al.,
where a crossover of the moduli is found at higher frequencies
at variance with bulk rheology. In any event, an overall reasonable agreement between the three sets of data is found,
which is remarkable taking into account that the results refer
to independent measures made in separate laboratories with
different techniques on different blood samples.

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.

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.

Adams BD, Baker R, Lopez JA, Spencer S (2010) Myeloproliferative
disorders and the hyperviscosity syndrome. Hematol Oncol Clin N
Am 24(3):585602
Alves MM, Rocha C, Goncalves MP (2013) Study of the rheological
behaviour of human blood using a controlled stress rheometer.
Clin Hemorheol Microcirc 53(4):36986
Ami RB, Barshtein G, Zeltser D, Goldberg Y, Shapira I, Roth A, Keren G,
Miller H, Prochorov V, Eldor A and others 2001 Parameters of red
blood cell aggregation as correlates of the inflammatory state. Am J
Physiol Heart Circ Physiol 280(5):H1982-H1988
Apostolidis AJ, Armstrong MJ, Beris AN (2015) Modeling of human
blood rheology in transient shear flows. J Rheol (1978present)
Apostolidis AJ, Beris AN (2014) Modeling of the blood rheology in
steady-state shear flows. J Rheol (1978present) 58(3):607633
Barnes HA (1997) Thixotropya review. J Non-Newtonian Fluid Mech
Baskurt O, Meiselman H (2003) Blood rheology and hemodynamics.
Semin Thromb Hemost 29(5):435450
Baskurt OK (2003) Pathophysiological significance of blood rheology.
Turk J Med Sci 33(6):347355
Baskurt OK (2007) Handbook of hemorheology and hemodynamics. IOS
Baumler H, Neu B, Donath E, Kiesewetter H (1999) Basic phenomena of
red blood cell rouleaux formation. Biorheology 36(56):439442
Bose S, Singh R, Hanewich-Hollatz M, Shen C, Lee C-H, Dorfman DM,
Karp JM, Karnik R (2013) Affinity flow fractionation of cells via
transient interactions with asymmetric molecular patterns. Sci Rep
3:2329. doi:10.1038/srep02329
Brust M, Aouane O, Thibaud M, Flormann D, Verdier C, Kaestner L,
Laschke MW, Selmi H, Benyoussef A, Podgorski T (2014) The
plasma protein fibrinogen stabilizes clusters of red blood cells in
microcapillary flows. Sci Rep 4:4348
Brust M, Schaefer C, Doerr R, Pan L, Garcia M, Arratia PE, Wagner C
(2013) Rheology of human blood plasma: viscoelastic versus
Newtonian behavior. Phys Rev Lett 110(7):078305
Bureau M, Healy JC, Bourgoin D, Joly M (1979) Etude rhologique en
rgime transitoire de quelques chantillons de sangs humains
artificiellement modifis. Rheol Acta 18(6):756768
Campo-Deao L, Dullens RP, Aarts DG, Pinho FT, Oliveira MS (2013)
Viscoelasticity of blood and viscoelastic blood analogues for use in
polydymethylsiloxane in vitro models of the circulatory system.
Biomicrofluidics 7(3):034102
Chien S (1970) Shear dependence of effective cell volume as a determinant of blood viscosity. Science 168(3934):977979
Chien S, King RG, Skalak R, Usami S, Copley AL (1975) Viscoelastic
properties of human blood and red cell suspensions. Biorheology
Chien S, Usami S, Dellenback R, Gregersen M (1967a) Blood viscosity:
influence of erythrocyte deformation. Science 157(3790):827829

Rheol Acta (2016) 55:485495

Chien S, Usami S, Dellenback R, Gregersen M, Nanninga L, Guest M
(1967b) Blood viscosity: influence of erythrocyte aggregation.
Science 157(3790):829831
Chien S, Usami S, Taylor HM, Lundberg JL, Gregersen MI (1966)
Effects of hematocrit and plasma proteins on human blood rheology
at low shear rates. J Appl Physiol 21(1):8187
Cho Y, Cho D, Rosenson R (2014) Endothelial shear stress and blood
viscosity in peripheral arterial disease. Curr Atheroscler Rep 16(4):
Daly A, Raval JS, Waters JH, Yazer MH, Kameneva MV (2014) Effect of
blood bank storage on the rheological properties of male and female
donor red blood cells. Clin Hemorheol Microcirc 56(4):337345
Datta SS, Gerrard DD, Rhodes TS, Mason TG, Weitz DA (2011)
Rheology of attractive emulsions. Phys Rev E 84(4):041404
Deplano V, Knapp Y, Bailly L, Bertrand E (2014) Flow of a blood analogue fluid in a compliant abdominal aortic aneurysm model: experimental modelling. J Biomech 47(6):12621269
Drasler WJ, Smith 2nd CM, Keller KH (1987) Viscoelasticity of packed
erythrocyte suspensions subjected to low amplitude oscillatory deformation. Biophys J 52(3):357365
Fabry TL (1987) Mechanism of erythrocyte aggregation and sedimentation. Blood 70(5):15721576
Fedosov DA, Pan W, Caswell B, Gompper G, Karniadakis GE (2011)
Predicting human blood viscosity in silico. Proc Natl Acad Sci
Guido S, Tomaiuolo G (2009) Microconfined flow behavior of red blood
cells in vitro. C R Physique 10:751763
Kohno M, Murakawa K, Yasunari K, Yokokawa K, Horio T, Kano H,
Minami M, Yoshikawa J (1997) Improvement of erythrocyte
deformability by cholesterol-lowering therapy with pravastatin in
hypercholesterolemic patients. Metab Clin Exp 46(3):287291
Kwaan HC, Wang J (2003) Hyperviscosity in polycythemia vera and
other red cell abnormalities. Semin Thromb Hemost 29(5):451458
Mason TG (2000) Estimating the viscoelastic moduli of complex fluids
using the generalized StokesEinstein equation. Rheol Acta 39(4):
McMillan DE (1976) Plasma protein changes, blood viscosity, and diabetic microangiopathy. Diabetes 25(2 SUPPL):858864
Merrill EW, Gilliland ER, Cokelet G, Shin H, Britten A, Wells RE (1963)
Rheology of human blood, near and at zero flow: effects of temperature and hematocrit level. Biophys J 3(3):199213
Merrill EW, Pelletier GA (1967) Viscosity of human blood: transition
from Newtonian to non-Newtonian. J Appl Physiol 23(2):178182
Picart C, Piau J-M, Galliard H, Carpentier P (1998) Human blood shear
yield stress and its hematocrit dependence. J Rheol (1978present)
Robertson A, Sequeira A, Owens R (2009) Rheological models for blood.
In: Quarteroni A, Veneziani A (eds) Formaggia L. Cardiovascular
mathematics Springer, Milan, pp. 211241
Sarkar S, Rosenkrantz TS (2008) Neonatal polycythemia and hyperviscosity. Semin Fetal Neonatal Med 13(4):248255
Shattil S, Furie B, Cohen H, Silverstein L, Glave P, Strauss M (2000)
Hematology: basic principles and practice. Churchill Livingstone,
Snabre P, Mills P (1996) II. Rheology of weakly flocculated suspensions
of viscoelastic particles. J Phys III 6(12):18351855
Sousa PC, Carneiro J, Vaz R, Cerejo A, Pinho FT, Alves MA, Oliveira
MS (2013) Shear viscosity and nonlinear behavior of whole blood
under large amplitude oscillatory shear. Biorheology 50(56):269
Steffen P, Verdier C, Wagner C (2013) Quantification of depletioninduced adhesion of red blood cells. Phys Rev Lett 110(1):018102
Thurston G, Henderson N (2006) Effects of flow geometry on blood
viscoelasticity. Biorheology 43(6):729746
Thurston GB (1972) Viscoelasticity of human blood. Biophys J 12(9):

Rheol Acta (2016) 55:485495

Thurston GB (1975) Elastic effects in pulsatile blood flow. Microvasc
Res 9(2):145157
Tomaiuolo G (2014) Biomechanical properties of red blood cells in health
and disease towards microfluidics. Biomicrofluidics 8(5):051501
Tomaiuolo G, Barra M, Preziosi V, Cassinese A, Rotoli B, Guido S
(2011) Microfluidics analysis of red blood cell membrane viscoelasticity. Lab Chip 11(3):449454
Tomaiuolo G, Guido S (2011) Start-up shape dynamics of red blood cells
in microcapillary flow. Microvasc Res 82(1):3541
Tomaiuolo G, Lanotte L, DApolito R, Cassinese A, Guido S 2015
Microconfined flow behavior of red blood cells. Med Eng Phys
2015 IPEM. Published by Elsevier Ltd.
Tomaiuolo G, Lanotte L, Ghigliotti G, Misbah C, Guido S (2012a) Red
blood cell clustering in Poiseuille microcapillary flow. Phys Fluids
Tomaiuolo G, Rossi D, Caserta S, Cesarelli M, Guido S (2012b)
Comparison of two flow-based imaging methods to measure

individual red blood cell area and volume. Cytom Part A 81(12):
Tomaiuolo G, Simeone M, Martinelli V, Rotoli B, Guido S (2009a) Red
blood cell deformability in microconfined shear flow. Soft Matter 5:
Tomaiuolo G, Simeone M, Martinelli V, Rotoli B, Guido S (2009b) Red
blood cell deformation in microconfined flow. Soft Matter 5(19):
Tran-Son-Tay R, Sutera SP, Rao PR (1984) Determination of red blood
cell membrane viscosity from rheoscopic observations of tanktreading motion. Biophys J 46(1):6572
Trappe V, Weitz DA (2000) Scaling of the viscoelasticity of weakly
attractive particles. Phys Rev Lett 85(2):449452
Yoon YZ, Kotar J, Yoon G, Cicuta P (2008) The nonlinear mechanical
response of the red blood cell. Phys Biol England, p. 036007
Zheng Y, Chen J, Cui T, Shehata N, Wang C, Sun Y (2014)
Characterization of red blood cell deformability change during
blood storage. Lab Chip 14(3):577583