Quarterly Journal of Experimental Physiology (1985) 70, 37-49

Printed in Great Britain

THE BLOOD RHEOLOGY OF MAN AND VARIOUS

ANIMAL SPECIES
T. M. AMIN AND J. A. SIRS
Department of Physiology and Biophysics, St Mary's Hospital Medical School, Paddington, London W2 IPG
(RECEIVED FOR PUBLICATION 23 MAY 1984)

SUMMARY

A comparative study has been made of the blood rheology, and its component factors, in horse,
sheep, cattle, goat, camel, pig, dog, rabbit and man. The erythrocyte flexibility of horse red
cells is high relative to man, that of pig, dog, camel and rabbit comparable, but less flexible,
and sheep, cattle and goat relatively inflexible. The erythrocyte flexibility of horse, sheep, cattle
and goats does not vary with the plasma fibrinogen level, as occurs with human and rabbit
cells. Washing erythrocytes and then suspending them in isotonic saline makes the erythrocytes
of all species relatively inflexible. There is a factor in horse plasma, which is not fibrinogen,
that makes horse and human erythrocytes suspended in it very flexible. The blood viscosity
of all species is comparable at high shear rates (230 s-') due to the shape of the cells
compensating for their flexibility. The variations of blood viscosity at low shear rates (11-5 s-1)
were also found to depend on the erythrocyte flexibility, and only influenced indirectly by the
fibrinogen concentration. There is no significant effect of temperature on the erythrocyte
flexibility of horse, sheep, cattle, goat and a small number of human subjects. This is reflected
in the way the viscosity of these bloods varies with temperature.
INTRODUCTION

It has been known for some time, following the work of de Haan (1918) and Fahraeus (1921),
that there are significant differences in erythrocyte aggregation, Rouleaux formation and
plasma proteins between animal species. A systematic investigation of the variation of blood
rheology, and in particular erythrocyte deformability, in species other than man has not
been made. There are indications that differences do exist (Ritcher, 1966), and nucleated
erythrocytes are believed to be less deformable than those of man. Since blood flow in the
circulation is basically the same in all mammals, this raises questions as to how blood flow
and oxygen transport could be adequately maintained, with wide variations of plasma
viscosity and erythrocyte deformability. These questions have a direct relevance to
pathological changes of blood rheology that occurs in human disease (Dintenfass, 1976).
There is considerable discussion and current interest in the significance of hyperviscosity
in man, and its contribution, either directly or indirectly, to the prognosis of disease, yet
it is possible that the degree of change would be accepted as Theologically normal in other
species.
Abnormalities of erythrocyte deformability, and associated blood rheology, in man, can
be classified into two main categories. The erythrocytes may be more flexible than normal,
which occurs in clinical situations such as bronchitis, some forms of hypertension, and in
the post-operative period (Dupont & Sirs, 1977), producing a lower viscosity at high shear
rates, but higher aggregation, Rouleaux formation and low shear rate viscosity. Alternatively,
there may be a decrease of erythrocyte deformability, such as occurs in sickle-cell anaemia,
iron deficiency anaemia and at low plasma fibrinogen levels, which increases whole blood
viscosity at high shear rates. The rate of oxygen exchange by inflexible erythrocytes is also
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T. M. AMIN AND J. A. SIRS

believed to be slower (Sirs, 1969). Yet man can be transfused stored blood with inflexible
cells (Sirs, 1966, 1969) relatively safely, and may be constantly subject 'in vivo' to
erythrocyte rigidity induced by the high osmotic concentrations during flow through the
vasa recta of the kidney. There are many unresolved questions as to the optimum rheological
behaviour of human blood that can be usefully considered in the context of the variations
that are present in other species.
METHODS

Human blood was obtained by venesection of the ante-cubital vein, whereas blood from other species
was obtained by puncture of the jugular vein. In all cases solid lithium heparin was used to prevent
coagulation at a concentration of 12 5 i.u. ml-' blood. All measurements were made within 6 h of
collecting the blood sample. The horse (thirty samples), sheep (ten samples) and cattle (ten samples)
bloods were obtained from the Wellcome Laboratories (Beckenham, Kent), and the goat (Golden
Gurnsey) and Bactrian camel bloods from the London Zoo Animal Hospital. In the case of the goat
(six samples) and camel (six samples), blood samples were always taken from the same healthy animal,
whereas the horse, sheep and cattle samples were obtained from various animals of the same species.
Multiple experiments were made on each blood sample.
Variation of the plasma fibrinogen level was obtained by diluting plasma with its own serum. The
serum was prepared by heating plasma at 56 0C for 10 min, then centrifuging at 1000 g for 30 min
to remove the fibrin clot. The fibrinogen concentration was estimated using the thrombin-clot
technique of Rampling & Gaffney (1976). At least two samples were used to obtain a mean, with
an error of +0 1 mg. ml-'.
Two methods were used to harden erythrocytes to varying degrees of inflexibility, without altering
their shape. The first was to incubate blood samples at 48 5 0C in a water bath, for varying time
intervals. The second was to add isotonic, pH = 7 4, formaldehyde solutions to blood samples, to
give mixed formaldehyde concentrations of 0 25, 0 5, 0 75 and 10%. The cells were checked
microscopically to ensure no change of shape occurred. The erythrocyte volumes (m.c.v.) were
measured by counting the cells in a 10 1dI sample, after dilution in 10 ml Isoton buffer solution, with
a Coulter Counter. The haematocrit was obtained by centrifugation on a Hawksley Microhaematocrit
centrifuge for 3 min. The m.c.v. was calculated by dividing the haematocrit by the erythrocyte count.
Both pH and tonicity were systematically monitored, the latter by measuring the freezing-point
depression.
The various human and horse proteins used in this study were obtained from Koch-Light
Laboratories. The proteins and their fractions were as follows: human and horse a-globulin (Cohn
fraction IV); human and horse f-globulin (Cohn fraction III); human and horse y-globulin (Cohn
fraction II); and human a,-globulin (Cohn fraction IV1). The proteins were dissolved in an isotonic
(pH 7 4) Ringer-Locke solution containing human serum albumin (Kabi) at a concentration of
2 g. 1-1. The concentration of each protein was made equal to that found in normal horse and human
plasma.
Rheological measurements of blood and plasma viscosities were principally made using a
Wells-Brookfield viscometer. At high shear rates, 230 s-1, the measurements have an accuracy of
+ 5%, which decreases to + 10% at 11 5 s-1. There were occasions, particularly with horse blood
which incurs errors in rotational viscometers due to an air-blood surface film, when independent
measurements of plasma and blood viscosities were made using a modified Coulter-Harkness capillary
method. The flexibility of the erythrocytes was ascertained by the centrifugal packing method of Sirs
(1970) using the stroboscopic recording method of Amin, Sirs & Turner (1983). A calibration curve
of the variation of the packing rate with haematocrit was obtained for each species and variation
of environmental factors, and the quoted packing rates have been corrected using these curves to a
standard haematocrit of 45 %. The small variation of erythrocyte and plasma specific gravities between
species has negligible effect on the packing rate.
RESULTS

Erythrocyte flexibility
The erythrocyte flexibility has been measured, using the centrifuge technique (Sirs, 1970),
in man, horse, sheep, cattle, pigs, goats, dogs, rabbits and camels. The data are summarized
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BLOOD RHEOLOGY

Table 1. Erythrocyteflexibility (% . min-'), m.c.v. (fi) and shape factor K,

for various animal species
Shape
factor,
K

Flexibility

Man
Horse

Sheep
Cattle
Goat
Camel
Pig
Dog

Rabbit

22 0C

30 OC

37 0C

M.c.v.

545
40-10
07
0-88
055
3-75
4 00
3 00
40

675
42 5
07
0-88
055
3 75

8
46 8
07
0 88
055
3 75

90
43
54
54
18
54
58
90
61

44 -

33
38
286
2-94
31
29

0
1o

4241!

Horse (0)

a
0

24
22
' 201

an()

68
0

2
Cattle (+)
0

vV

4
1

Sheep (v)

v
3

Plasma fibrinogen concentration (mg ml-')

Fig. 1. The variation of erythrocyte flexibility (% .min-'; at haematocrit 45%) with plasma fibrinogen
concentration (mg. ml-') at 30 'C, for 0, horse; *, man; V, sheep; and +, cattle.

in Table 1. The packing rates for rabbits, pigs, camels and dogs are comparable, though
less than man; the erythrocytes of sheep, cattle and goats are less flexible, while those of
horses are very flexible. Rampling & Sirs (1972) observed that the flexibility of human
erythrocytes depends on the plasma fibrinogen concentration. This is also true of rabbit
cells (Sirs & MacDonald, 1974). In contrast, neither flexible horse erythrocytes nor the
erythrocytes of sheep, cattle, goats or camel blood are modified by fibrinogen, as indicated
in Fig. 1. The packing rate of horse erythrocytes is only slightly lowered by suspension in
horse serum, relative to suspension in their normal plasma. However, a large decrease of
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T. M. AMIN AND J. A. SIRS

(%0 . min-') of resuspending erythrocytes in the

plasma of a different species

Table 2. The effect on erythrocyte flexibility

Human erythrocytes
Horse erythrocytes

Sheep erythrocytes
Cattle erythrocytes

Human plasma
(fibrinogen
2 68 mg. ml-')

Horse plasma
(5 5 mg. ml-')

Sheep plasma
(3 6 mg. ml-')

Cattle plasma
(3-9 mg. ml-')

6 75
33
0-63
0-65

31-5
42-5
09
0-89

Haemolysis
Haemolysis
07
0-83

Haemolysis
Haemolysis
0-67
0-88

cell flexibility occurs if horse erythrocytes are repeatedly washed in isotonic saline, or
Ringer-Locke solution, then resuspended in saline. The erythrocyte flexibilities for cells in
saline suspensions at 30 0C and a haematocrit of 450, are 1 2, 4 0, 0-6, 0-85, 0-69 and
1 9500% . minI for man, horse, sheep, cattle, goat and camel respectively. Relative to the other
species, horse erythrocytes still retain a higher flexibility in saline. Simply diluting horse
plasma with Ringer-Locke solution also progressively decreases the flexibility of horse cells
suspended in the mixed solution.
The horse plasma factor
Experiments were then undertaken to ascertain the effect of suspending the cells of one
species in the plasma of another species. Checks were made before these investigations to
avoid antigen-antibody reactions, by the following procedures. A volume of washed
erythrocytes from one species was added to an equal volume of plasma from another species.
The suspension was mixed, then left for 20 min before reseparating the plasma. The
procedure was repeated a second and third time, with fresh-washed cells, to ensure complete
removal of any antibody that reacted with the cells. A further suspension of fresh-washed
cells was prepared, and a sample from it checked by microscopic examination for the
presence of cell agglutination. The following tests were only made if no agglutination was
observed. If, after this washing procedure, the cells are resuspended in the plasma of
different species, the erythrocyte flexibilities are modified to those shown in Table 2.
Particularly significant is the large increase in packing rate of human cells in horse plasma,
and the equivalent high rate retained by horse cells in human plasma. Suspending horse
cells in human serum, and human cells in horse serum, gave packing rates of 33 4 and
15.500 min- respectively, so this effect is not due specifically to fibrinogen. Though no effect
of human fibrinogen concentration was found for horse cells in human plasma, the packing
rate for human cells in horse plasma is doubled if the horse fibrinogen concentration is raised
above a threshold level of about 2-4 mg. ml-', to the value given in Table 2.
Preliminary investigations have been undertaken to identify the factor in horse plasma
that so dramatically modifies human erythrocyte flexibility. In the first of these experiments,
human and horse plasma or serum was dialysed in size 18/32" Visking tubing, with a
molecular-weight cut-off of 5000, against saline or Ringer-Locke solution at pH 7-4 for at
least 4 h. The flexibility of horse or human erythrocytes in dialysed plasma was not changed
relative to suspension in undialysed plasma or serum. This led to an investigation of the
effect of the addition of horse and human a-, ,-, and y-globulins, and human a,-globulins,
to suspensions of human and horse cells in plasma, serum, Ringer-Locke solution with
albumin and fibrinogen, and Ringer-Locke with albumin solution. The possibility that the
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BLOOD RHEOLOGY

41

Table 3. The percentage change of horse and human erythrocyte flexibility, relative to
control samples, following the addition of horse ax-, /3- and y-globulins
Horse proteins
Horse cells in plasma
Horse cells in serum
Horse cells in Ringer-Locke-albumin/
soln.
Human cells in Ringer-Locke-albumin-/
fibrinogen soln.
Human cells in Ringer-Locke-albumin/
soln.

a+,

a+y

/1+y

+ 14
+ 13

+ 16
+ 15

-18

-24

+42
+ 37
+ 50

-36

-38

-13

-79

-4

+ 50

-31

-14

+ 114

-7

-20

+ 21

-40

-45

-22

factor might be a protein was partly suggested by similar effects that have been observed
when erythrocytes from diabetic patients are placed in normal plasma. A direct comparison
was made of the packing rates between suspensions of cells with and without the addition
of protein. There were only two occasions where human proteins produced a significant
effect on human cells. With Ringer-Locke-fibrinogen-albumin suspensions, the presence
of a1-globulin decreased the flexibility from 8-5 in the control sample to 2.9% . min-. In
Ringer-Locke-albumin solution without fibrinogen, y-globulin slightly increased the
flexibility from 2-8 in the control sample to 4% . min'. There were no changes relative to
control samples for horse cells suspended in human protein solutions. The relative change
of erythrocyte flexibility, following the addition of horse plasma proteins to horse and
human erythrocytes in plasma, serum and Ringer-Locke solution, is shown in Table 3.
Horse erythrocytes in plasma show an increase in flexibility following addition of ax-, /and y-globulins. The relative changes in serum were similar. Within experimental errors,
a-, ,- and y-globulins, as well as combinations a +,/, a + y and / + y, had no significant
effect on horse cells in Ringer-Locke with human serum albumin solutions. For human
erythrocytes in Ringer-Locke-fibrinogen-albumin solutions, f-globulins had no significant
effect, a-globulins reduced the flexibility, and y-globulins increased the flexibility. For
combinations of globulins, a with /3 reduced the flexibility, a with y reduced it slightly, but
, with y increased the flexibility by a factor of more than two. In the absence of fibrinogen
in Ringer-Locke-albumin solution, the addition of a-, ,- and y-globulins, and combinations
of these globulins, had only a small effect on human erythrocyte flexibility. These changes
are, however, small relative to the effect of suspending human cells in horse plasma.
Blood viscosity
A systematic investigation of blood viscosity at shear rates from 230 to 11 5 s-1 was
undertaken using a Wells-Brookfield viscometer and a Coulter-Harkness capillary
viscometer, with blood from horses as representative of a species with very flexible cells,
from man for the intermediate range, and from sheep and cows for blood with relatively
inflexible erythrocytes. The variation of blood viscosity with shear rate, at 37 3 0C, corrected
to a haematocrit of 30 , is shown in Fig. 2. Compared to the change of erythrocyte
flexibility, the blood viscosity at high shear rates for the different species is surprisingly close.
It was initially considered possible that the higher blood viscosity of horse, relative to man,
was due to some aggregation still being present at 230 sol. This was resolved by
measurements of horse and human blood viscosities using the Coulter-Harkness technique
at flow rates with a mean shear rate of 450 s-l, with the same results. The effect of varying
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T. M. AMIN AND J. A. SIRS

42

6
0
0
0

30 115 23

46

115

230

Shear rate, T (s-l)

Fig. 2. The effect of shear rate (s'l) on whole blood viscosity at 37 5 'C and haematocrit 30%, for +, man; x,
horse; 0, sheep; 0. cattle; A, goat; and V, camel.

3-6,

3.4
A,
k
o
o

32;

0~

3 0-

ok

28-

I.

oo (0)
Horse

Cattle (+)
Sheep (v)

+ ; +

~
28
Human (-)

26

Plasma fibrinogen concentration (mg. ml-')

Fig. 3. The variation of intrinsic viscosity, KT, with fibrinogen concentrations at 30 'C, for 0. horse; +, cattle;

V, sheep; and 0, man. The values of KT were calculated from the slope of plots of the natural logarithm of
the relative viscosity against haematocrit, at each fibrinogen concentration and a shear rate of 230 s'l. Note
the similar invariance of the erythrocyte flexibility with fibrinogen concentration, for horse, sheep and cattle
in Fig. 1.

plasma fibrinogen by replacing plasma with serum is shown in Fig. 3. As has been observed
previously (Dupont & Sirs, 1979), the viscosity of human blood is sensibly constant with
variation of the plasma fibrinogen level. The increase of plasma viscosity as the plasma
fibrinogen concentration rises is balanced by the increase of flexibility shown in Fig. 1. With
horse, sheep and cattle however, the blood viscosity rises with the fibrinogen level, consistent
with the invariance of their cell flexibility indicated in Fig. 1.
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BLOOD RHEOLOGY

1.0

0.8'0

Q,

0-6

00-40-

040

50
30
40
60
Haematocrit (%)
Fig. 4. Typical plots, at a shear rate of 230 s'l and 30 'C, of the natural logarithm of the relative viscosity against
haematocrit, for 0, horse, with a fibrinogen concentration equal to 3-8 mg. ml-'; V, sheep, 3 6 mg ml-'; +,
cattle, 3 3 mg. ml-'; and 0, man, 2 68 mg. ml-'. The dashed line, for man, demonstrates the increased slope
that occurs at a lower shear rate of 23 s'l. The slope of the lines at high shear rates is equal to KT. More than
ninety plots of In Yr against H were obtained during these investigations, and on only three occasions did the
correlation coefficient fall below 0 75.
10

20

The erythrocyte shape factor, K

An appreciation of why the variation of blood viscosity at high shear rates between species
is so small can be obtained by plotting the natural logarithm of the relative viscosity (i.e.
blood viscosity/plasma viscosity) against haematocrit, as shown in Fig. 4. The slope of these
lines is determined by two factors, the shape and flexibility of erythrocytes. Unfortunately
it is not possible to assess which factor has changed solely from these lines. The shape factor
can, however, be estimated in the following way. The linear relation can be expressed
mathematically as:
(1)
In Yb - In = aH,

yp

where H is the haematocrit, a the slope of the line, Yb the blood viscosity and yp the plasma
viscosity. Rearranging eqn. (1), by replacing the ratio Yb/Yqp by the relative viscosity Yr, we
(2)
obtain:
(aH) = I + aH+ (aH)2/2 +...,

Yrexp

and when H is less than 0-01:

Yr

I+ aH.

(3)

A theoretical analysis of the viscosity of a dilute suspension of rigid spheres was first derived
by Einstein (1906). Later Jeffery (1922) extended the theory to rigid ellipsoidal shapes. An
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T. M. AMIN AND J. A. SIRS

allowance for the internal viscosity of immiscible spheres on the suspension viscosity was
developed by Taylor (1932). These equations can be put in the general form:
(4)
Yr= l+KTH,
where K is the shape factor and T is equivalent to Taylor's factor to allow for the internal

viscosity of the fluid drop. If the internal viscosity is high, and the drop effectively rigid,
T becomes equal to 1. Putting K equal to 2 5, with T= I in eqn. (4), gives Einstein's
equation for a dilute suspension of rigid spheres. For asymmetric shapes K is greater than
2 5, and may be as high as 40 for a long rod-like structure, such as the fibrinogen molecule.
If the immiscible fluid drop has an internal viscosity just higher than the supernatant fluid,
like erythrocytes in plasma, Tis less than unity, and Yr is correspondingly smaller. In practice
K varies as the cells are deformed, and the extent of deformation will be related to T, so
the two components interact. By comparison of eqns. (3) and (4), it can be seen that 'a'
is equal to KT. Since a is a constant, this equivalence, obtained strictly at low H, will apply
for all H in Fig. 4. The value of KT, at high shear rates with no aggregation between the
cells, can be obtained from the slope of the line. It can be obtained less accurately on a
single blood sample of known H by measuring Yb and p then calculating In (Yb!Yp)/H. The
differences between the values of KT for the different species, and the direct measurements
of flexibility using the centrifuge technique, imply that the shape factor K must vary from
one species to another. To confirm this deduction, estimates have been made of K by making
erythrocytes inflexible, so that T is equal to 1 and the packing rate is effectively zero. With
human erythrocytes it is possible to decrease progressively the erythrocyte flexibility by
incubating the blood at 48 5 'C for increasing lengths of time, or by adding weak
formaldehyde solutions. These procedures do not appear to alter the cell volume or shape
(Sirs, 1981). Measurements were made of the haematocrit, packing rate and relative
viscosity after heating human blood for 5, 10, 15 and 20 min. The value of KT was then
plotted against the packing rate as shown in Fig. 5. This is linear with a correlation
coefficient of 082, and from the intercept with the y-axis, when the cells are inflexible, the
value of K can be estimated as 3-26 + 007. With sheep and cattle erythrocytes, which are
naturally almost inflexible, an estimate of the value of K can be made directly from the
normal blood and plasma viscosities. Various attempts to make these cells even less flexible
had no effect on the magnitude of K. The shape factor for horse red blood cells proved
the most difficult to estimate. Heating at 48 5 'C does not induce rigidity with these cells,
and adding agents such as formaldehyde also agglutinates plasma proteins. We were only
able to provide an estimate of the shape factor for horse cells by repeated washing in saline
followed by resuspension in 2% formaldehyde in Ringer-Locke solution, before measuring
the relative viscosities and packing rate. A summary of the Theological parameters and
estimates of the shape factor K, for man, horse, sheep and cattle is shown in Table 1. It
demonstrates why the viscosity of cattle and sheep blood, with relatively inflexible
erythrocytes, is still comparable to human blood at 37-5 0C and the same haematocrit,
because the shape factors at 2-84 and 2 94 are lower than for man, at 3 26, and compensate.
By contrast the shape factor for horse is higher, at 3-8, which offsets the high flexibility of
these erythrocytes.
The influence of erythrocyteflexibility on Rouleaux formation
At a shear rate of 11 5 s'l, sheep and cattle bloods are less viscous than man, and in turn
horse, as shown in Fig. 2. This is the reverse situation to that pertaining at high shear rates.
A decrease of erythrocyte flexibility increases blood viscosity at high shear rates (Chien,

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BLOOD RHEOLOGY

4-0

k3-5

CA

, 3030
2-5
2-0-

10

11

Erythrocyte flexibility index (% . min-')

Fig. 5. The variation of erythrocyte flexibility, measured by the centrifuge technique, against intrinsic viscosity,
KT, measured using a Wells-Brookfield viscometer at 230 s-l, at 30 'C; 0, normal erythrocytes; 0, heathardened cells; A, formaldehyde-fixed cells. The line was fitted by the method of least squares, with a correlation
coefficient of 0-82. The bars represent the error in KT of +0-2, due to the experimental errors inherent in
measuring blood and plasma viscosities.

Luse, Jan, Usami, Miller & Fremont, 1967), and a corresponding increase would be
expected at low shear rates. The additional component that influences viscosity at low shear
rates is aggregation and Rouleaux formation. Rouleaux formation is absent in the blood
of sheep and cattle, but high in horses (de Haan, 1918). It is also often stated that Rouleaux
formation, and in turn low shear rate viscosity, is directly related to the fibrinogen
concentration. To elucidate further how erythrocyte flexibility influences blood viscosity and
aggregation, measurements were made of the relative viscosity of human blood at a
haematocrit of 4500, with suspension of the cells in plasma, serum and Ringer-Locke
solution, and alteration of the flexibility using the techniques discussed above. The flexibility
of each sample was assessed using the centrifuge method, and the corresponding blood and
plasma viscosities at 37 5 0C over a range of shear rates from 11 5 to 230 s-l. The variation
of viscosity with erythrocyte flexibility is shown in Fig. 6. At high shear rates the relative
blood viscosity increases with decreasing flexibility as expected. At low shear rates, of 23
and 11 5 s-1, the blood viscosity falls initially, before increasing as the cells become less
flexible. There is no association, at low shear rates, of these changes of viscosity with
fibrinogen concentration in plasma with fibrinogen, and in serum and Ringer-Locke
solution without fibrinogen. At high shear rates the increase in viscosity is consistent with
the increased rigidity of the cells, in accord with eqn. (4). At low shear rates the relative
viscosity depends on aggregation, as well as shape and cell flexibility. Decreasing flexibility
should, as at high shear rates, increase the viscosity, but this is offset and reversed by an
associated fall of aggregation. The latter effect is consistent with the difference of blood
viscosity between horse, sheep, cattle and man at low shear rates. Whether the viscosity
at low shear rates increases or decreases as the erythrocytes are hardened depends on the
initial flexibility and aggregation. With horse and human cells the flexibility and aggregation
are relatively high, so a decrease of blood viscosity is observed when the erythrocyte
flexibility is decreased. With cattle and sheep, where the cells are already less flexible and
negligible Rouleaux formation occurs, decreasing the flexibility leads to an increase of
viscosity at all shear rates.

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T. M. AMIN AND J. A. SIRS

46

12
6

T=230s-10

=23 s'

115 s-1
0

..; Erythrocyte

101
~~~~~~~61

6-T
2

o.

flexibility index (%b. min1)

~~~~~~~16

Eiythrocyte flexibility index

(to . mind

Fig. 6. The effect of human erythrocyte flexibility on the relative viscosity at shear rates, T, of 11 5-230 s' at
375 C and at a haematocrit of 45% . The fibrinogen concentration was varied by suspending erythrocytes in:
*, plasma; 0. serum and A, Ringer-Locke solution, without modifying the interrelation of flexibility to
relative viscosity.

The changes of blood rheology with temperature

The variation of blood viscosity with temperature between human individuals and
different animal species is also dependent in a similar manner on erythrocyte flexibility and
aggregation. In the majority of healthy human subjects erythrocyte flexibility decreases as
the temperature falls. There are some human subjects with a packing rate that is unaffected
by a decrease of temperature. The erythrocyte flexibilities of horse, sheep and cattle are also
relatively unaffected by temperature, as shown in Table 1. Similar measurements of relative
viscosity at a constant haematocrit, using the Coulter-Harkness viscometer, confirmed that
it was constant with temperature, consistent with the invariance of flexibility with
temperature. Measurements of the effect of temperature on the relative viscosity of blood
at high and low shear rates disclosed a significant difference in the behaviour of bloods
containing flexible cells that did not alter with temperature. In this case there is an increase
of aggregation and Rouleaux formation as the temperature falls. With horse blood, and
that of humans with flexible and temperature-invariant cells, there is a particularly large
increase of blood viscosity at low shear rates and a shift of the anomalous behaviour of
blood to higher shear rates, as indicated in Fig. 7. With cattle and sheep, where there is
low or negligible aggregation, there is only a moderate rise in viscosity at shear rates from
11 *5 to 230 s'l, consistent with the increase of viscosity normally expected at a lower
temperature. In the majority of human bloods, the fall of erythrocyte flexibility with
temperature offsets the tendency for Rouleaux to increase, and there is a correspondingly
smaller rise in blood viscosity at low shear rates.

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BLOOD RHEOLOGY

8-

6-

W4)
--+--'

-----

2-

200
100
Shear rate (01)
Fig. 7. A comparison of the effect of temperature on the dependence of blood viscosity on shear rate; for 0,
horse, representative of blood with very flexible cells; and +, cattle, representative of blood with relatively
inflexible cells. The continuous curves are for measurements made at 20 0 0C, and the dashed curves at 37 5 0C,
with a haematocrit of 3100.
0

DISCUSSION

Though erythrocyte flexibility varies significantly with species, it is initially surprising how
small the corresponding differences in whole blood viscosity are at high shear rates. The
experimental data suggest that there is a balance between plasma viscosity, erythrocyte
flexibility and cell shape, which contribute towards maintaining blood viscosity for a given
haematocrit within a fairly limited range. Sheep and cattle have relatively inflexible cells,
which in humans would give rise to a pathologically high viscosity, that is offset by a
decrease of the shape factor, K, so only a small increase of viscosity results. The converse
applies to horse cells.
At low shear rates there is also an association of blood viscosity and erythrocyte
flexibility. This is consistent with flexibility being related to aggregation and Rouleaux
formation. The very high flexibility of horse erythrocytes will facilitate capillary blood flow
and oxygen exchange, but predispose towards aggregation at low shear rates in large veins.
An increase of flexibility is observed in man, though still less than in horse, in various
pathological states such as bronchitis, hypertension and during the post-operative period.
It is therefore of interest why the horse is not subject to an increased risk of venous
thrombosis. The answer appears to be that at rest, and lower blood flow, the horse has the
ability to lower its haematocrit to 30-35% by sequestration of the cells in the spleen. The
lower haematocrit has a greater effect on blood viscosity at low shear rates, as can be
appreciated from Fig. 4. Just before activity and higher blood flow, the horse can increase
its haematocrit to 450 by contraction of the spleen. The horse has apparently adapted its
blood rheology to obtain maximum flow and oxygen transport, while reducing the
associated problems of aggregation and sludging. The situation appears to be critically
balanced however, recent studies suggesting that navicular disease in the horse (Sirs, Amin,
Coles & Allen, 1984) is associated with a small increase of erythrocyte flexibility producing
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T. M. AMIN AND J. A. SIRS

sufficient additional aggregation to adversely reduce blood flow to the navicular bone. The
high flexibility of horse cells appears to be associated with a factor in plasma that is adsorbed
on to the cell surface. The factor is not fibrinogen, can be washed easily from the cell surface,
and the cell flexibility depends on its concentration. The dialysis experiments indicate that
it has a molecular weight greater than 5000. Though effects were observed on the addition
of a-, #- and y-globulins to cell suspensions, and there is some synergystic action between
these proteins, no one factor has yet been identified that can produce the striking difference
between cells in Ringer-Locke solution and serum. The action of the factor in horse serum
on human cells has a potential therapeutic value.
The over-all contribution of erythrocyte flexibility to blood viscosity is best summarized
by the lines in Fig. 4. At high shear rates the slope of the line is determined by the shape
factor, K, and the erythrocyte flexibility. At low shear rates an additional factor must be
included to allow for the energy dissipated in breaking down Rouleaux and the change of
particle shape due to aggregation. Blood with very flexible cells has a less-steep plot of
natural logarithm of relative viscosity against haematocrit at high shear rates, and at low
shear rates, due to increased aggregation, a higher slope. In man this produces the surprising
observation that if the erythrocyte flexibility is reduced, the high shear rate viscosity may
increase, while the low shear rate viscosity decreases. This critically depends on the
erythrocyte flexibility, if this is low initially, as in sheep and cattle a decrease of flexibility
increases blood viscosity at both high and low shear rates, because the contribution of
aggregation to viscosity is small. In man, erythrocyte flexibility is related to the plasma
fibrinogen level (Rampling & Sirs, 1972). No similar action was observed in sheep, cattle
or horse, though it is known to affect rabbit cells. The low shear rate viscosity, at 11 -5 s-',
is likewise not significantly altered in sheep, cattle or horses by fibrinogen. Modifying
erythrocyte flexibility by suspension of the cells in plasma, serum and Ringer-Locke
solution, or by heating at 48 0C, alters low shear rate viscosity. The changes shown in Fig. 6
correlate with cell flexibility and appear otherwise independent of the fibrinogen level.
This casts doubt on the accepted hypothesis that fibrinogen binds to erythrocytes and forms
a bridge between cells resulting in Rouleaux. An alternative explanation is that fibrinogen
acts indirectly, by modifying erythrocyte flexibility, and its presence is not an essential factor
in determining the anomalous viscosity of blood at low shear rates. How proteins, such as
fibrinogen, influence erythrocyte flexibility is not established. Fibrinogen is adsorbed on to
the cell surface, as are other plasma proteins (Gramlich, 1963), and is a component of the
buffy coat that provides a micro-environment adjacent to the cell. By its nature adsorption
will lower the Gibb's free energy and provide an environment that will facilitate shape
changes. Fibrinogen may contribute more to this reduction of energy than other proteins,
because of its long flexible structure with actin-myosin-like properties. Aggregation between
cells can be envisaged as being determined by the lower free energy obtained by fusing cells
together, akin to immiscible oil drops in water, and the degree to which the energy falls
is dependent on the shape changing to give maximum contact. It has been shown by electron
microscopy that Rouleaux formation involves a change of shape, and the biconcave surfaces
are flattened in the region of contact (Chien et al. 1971; Rowlands & Skibo, 1972). The
phenomenon is however multifactorial, as the previous results demonstrate, and the
contribution of any one factor critically depends on the balance of all the components.
This is particularly the case in explaining the effect of temperature on blood viscosity.
While with simpler fluids, such as plasma, a decrease of temperature increases viscosity by
about 2-3% . C-1C, its effect on blood viscosity is complex. It particularly depends on
erythrocyte flexibility and aggregation. It would appear that there are a number of human
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49

subjects whose erythrocyte flexibility does not fall significantly with temperature. In these
circumstances the low shear rate viscosity increases rapidly with hypothermia, due to
increased aggregation. Horses are subject to the same problem. The results of varying
temperature again underline the primary role of erythrocyte flexibility and haematocrit in
low shear rate viscosity.
We are indebted to the Wellcome Laboratories, Kent, and the Regents Park Zoo Animal Hospital,
London, for the supply of animal bloods.
REFERENCES

AMIN, T. M., SIRS, J. A. & TURNER, P. (1983). Measurement of erythrocyte deformability using a
stroboscopic recording centrifuge. Physics in Medicine and Biology 28, 269-275.
CHIEN, S., LUSE, S. A., JAN, K. M., USAMI, S., MILLER, L. H. & FREMONT, H. (1971). Effects of
macromolecules on the rheology and ultrastructure of red cell suspensions. In Proceedings of the
Sixth European Conference on Microcirculation, pp. 29-34. Basel: Kager.
CHIEN, S., USAMI, S., DELLENBACK, R. J. & GREGERSON, M. I. (1967). Blood Viscosity: Influence
of erythrocyte deformation. Science 157, 827-829.
DE HAAN, J. (1918). Uber die Senkungsgeschwindigkeit der Blutkorperchen verscheidener Blutarten
im Hinblick auf deren Verwendbarkeit fur Phagocytoseuntersuchungen. Biochemische Zeitschrift
86, 298-308.
DINTENFASS, L. (1976). Rheology of Blood in Diagnostic and Preventive Medicine. London:
Butterworths.
DUPONT, P. A. & SIRS, J. A. (1977). The relationship of plasma fibrinogen, erythrocyte flexibility and
blood viscosity. Thrombosis and Haemostasis 38, 660-667.
EINSTEIN, A. (1906). Eine neue Bestimmung der Molekuldimensionen. Annalen der Physik 19, 289-306.
FAHRAEUS, R. (1921). The suspension stability of blood. Acta medica scandinavica 55, 1.
GRAMLICH, F. (1963). Die Bindungsfahigkeit roter Blutkorpen. Folia haematolia, Neue Folge 9, 15-20.
JEFFERY, G. B. (1922). The motion of ellipsoidal particles immersed in a viscous fluid. Proceedings
of the Royal Society A102, 161-179.
RAMPLING, M. W. & GAFFNEY, P. J. (1976). The sulphite precipitation method for fibrinogen
measurement; its use on small samples in the presence of fibrinogen degradation products. Clinica
chimica acta 67, 43-52.
RAMPLING, M. W. & SIRS, J. A. (1972). The interaction of fibrinogen and dextrans with erythrocytes.
Journal of Physiology 223, 199-212.
RITCHER, W. (1966). Relative aggregation tendency of erythrocytes from man and various animal
species. Acta chirurgica scandinavica 132, 601-612.
ROWLANDS, S. & SKIBO, L. (1972). The morphology of red cell aggregates. Thrombosis Research 1,
47-58.
SIRS, J. A. (1966). Discussion. In Hemorheology: Proceedings of the First International Conference, ed.
COPLEY, A. L., p. 477. Oxford: Pergamon Press.
SIRS, J. A. (1969). The respiratory efficiency and flexibility of erythrocytes stored in acid-citratedextrose solution. Journal of Physiology 203, 93-109.
SIRS, J. A. (1970). Automatic recording of the rate of packing of erythrocytes in blood by a centrifuge.
Physics in Medicine and Biology 15, 9-14.
SIRS, J. A. (1981). Erythrocyte flexibility and whole blood viscosity. In Clinical Aspects of Blornd
Viscosity and Cell Deformability, ed. LOWE, J. D. O., BARBENEL, J. C. & FORBES, C. D., pp. 9-18.
Berlin: Springer-Verlag.
SIRS, J. A., AMIN, T., COLLES, C. M. & ALLEN, B. (1984). The effect of warfarin on blood rheology
in horses. Proceedings of the Association for Veterinary Clinical Pharmacology and Therapeutics 8,
109-112.
SIRS, J. A. & MACDONALD, G. J. (1974). Changes of erythrocyte flexibility during and following
surgery on rabbits. Thrombosis Research 5, 657-666.
TAYLOR, G. I. (1932). The viscosity of a fluid containing small drops of another fluid. Proceedings
of the Royal Society A138, 41-48.

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