Mechanism of Erythrocyte Aggregation and Sedimentation

By Thomas L. Fabry

Unstirred suspensions of erythrocytes form stable spheri- tor of anion transport, but not by ouabain, a cation trans-
cal aggregates of uniform size. The radius of the spheres port inhibitor. The kinetics of erythrocyte sedimentation
depends upon the suspending medium and the hematocrit. reflects the aforementioned mechanism: no sedimentation
Erythrocyte suspensions will undergo sedimentation only occurs during rouleau formation. Once the spheres of
after these aggregates are formed. Aggregation is a two- uniform size are formed, they will settle according to the
step process: first. erythrocytes associate in long chains Einstein-Stokes equation. In this model, parameters of
(rouleau formation). Next, these chains form spheres of sedimentation kinetics are the delay before sedimentation
uniform size. The requirements for this well-defined pro- starts, the rate of sedimentation in the steady state, and
cess are an electrolyte and a neutral or negatively charged the radius of the sedimenting aggregate. The radius can be
macromolecule in the solution and a metabolically active calculated from the rate of fall of the aggregates and
red cell. If these conditions are not met, red cells either will agrees well with the microscopically observed radius. It is
not aggregate at all or will form amorphous aggregates. inversely proportional to the hematocrit, which explains
Rouleau formation and sedimentation are inhibited by the elevated sedimentation rates in anemia.
4,4’-diisothiocyanatostilbene-2,2’-disulfonic acid, an inhibi- C 1987 by Grune & Stratton, Inc.

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T HE
(ESR)
ERYTHROCYTE
is a useful qualitative
SEDIMENTATION
empirical
RATE
index of nonspe-
possible by developing
ing mechanism.
a kinetic model
This may make erythrocyte
based on the underly-
sedimentation a
cific disease activity.’ Its clinical usefulness is, however, useful tool for investigating the pathophysiology of some
limited by three major problems: (a) quantitative interpreta- diseases. Furthermore, quantitative interpretation of sedi-
tion is not possible, (b) there is a poor correlation with mentation may provide an inexpensive screening test of
measurements of other acute phase reactants, and (c) it is plasma protein and erythrocyte membrane abnormalities in
paradoxically low in several diseases (eg, certain forms of some less common disorders.
inflammatory bowel disease,2 sickle cell disease,3 and possi-
bly in congestive heart failure4). Increased ESR has been MATERIALS AND METHODS
attributed to increased amounts of some plasma proteins
Blood specimens anticoagulated with K3EDTA were obtained
(fibrinogen,5 etc), but again there is only very poor quantita-
from healthy volunteers and were processed immediately after
tive correlation. Expressing the sedimentation rate as the
collection. Red cells were suspended in autologous plasma to obtain
amount of sedimentation at one hour is only meaningful if different final hematocrit values (Hct). For the inhibition experi-
the process occurs uniformly over time. Any deviation from ments, washed red cells were incubated with 4,4’-diisothiocyanato-
zero-order kinetics means that more than one parameter stilbene-2,2’-disulfonic acid (DIDS) (0.1 mg/mL) for 30 minutes at
must be used to describe the process. 37#{176}C. This is the same concentration of DIDS that is used to inhibit
Chien6 laid the foundation for interpreting the rheology of the chloride/bicarbonate antiporter of the erythrocyte membrane.
red cell suspensions. He established the need for electrolytes After incubation the cells were washed with isotonic saline contain-
and macromolecules to be present in the suspending medium ing 10 mmol/L glucose and then reconstituted with plasma.
Ouabain (Sigma Chemical Co, St Louis) was dissolved in water
for rouleau formation to occur. Perelson and Wiegel,7 using
(0.01 mol/L stock solution; final concentration of incubation mix-
techniques of statistical mechanics, showed that the average
tures, 0.1 mmol/L). DIDS was prepared in HEPES buffer immedi-
rouleau size increases as the adhesion energy between eryth-
ately before use. Other chemicals were obtained from standard
rocytes increases. Samsel and Perelsont developed an elegant sources.
kinetic model for rouleau formation and pointed out that as Sedimentation experiments were performed in Kimble disposable
rouleaux grow large they form rings and branching aggre- 200-mm Westergren tubes at pH 7.4 and at 25 ± 2#{176}C. Experiments
gates. Evans and Buxbaum9 and Buxbaum et al’0 quantified were completed within three hours of sample preparation to avoid
the surface affinities of erythrocyte membranes for macro- artifacts from irreversible aggregation of red cells. To obtain the
molecules and related them to aggregation. photomicrographs, erythrocyte suspensions were allowed to sedi-
Quantitative interpretation of the sedimentation rate is ment in rectangular cross-section tubes 0.2 x 5 or 0.3 x 5 mm and
100 mm long (Vitro Dynamics, Rockaway, NJ). The kinetics of
sedimentation in this tube is the same as in a Westergren tube filled
to 100 mm. The tubes were placed flat side down on the microscope
From the Department of Medicine, Division of Gastroenterology.
stage in different phases of the sedimentation, and photographs were
The Mount Sinai School of Medicine and The Mount Sinai
taken at 4x and lOx magnification.
Medical Center, New York.
Curve fitting and statistical calculations were carried out by using
Submitted May 2. 1 986; accepted July /3, 1987.
the computer programs SAS” and Statgraphics.’2 The Einstein-
Address reprint requests to Thomas L. Fabry. MD, PhD, Depart-
Stokes equation was used as derived by Hiemenz.’3
ment ofMedicine. Mount Sinai School ofMedicine and The Mount
Sinai Medical Center, New York, NY 10029.
The publication costs ofthis article were defrayed in part by page RESULTS
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with /8 U.S.C. §1734 solely to Kinetics of erythrocyte sedimentation and the effect of
indicate this fact. Hct. Erythrocyte sedimentation follows an S-shaped curve
© 1987 by Grune & Stratton, Inc. with time (Fig 1 and Table I ). There is a delay in the start of
0006-4971/87/7005-0045$3.00/0 sedimentation; during this time the rate of sedimentation is

1572 Blood, Vol 70, No 5 (November), 1987: pp 1572-1576

ERVTHROCVTE AGGREGATION AND SEDIMENTATION 1573

Table 2. Effect of Anion and Cation Transport Inhibitors

on Sedimentation
E
Concentration
E
Inhibitor (iunol/L) Slope ESR
z
0
Blank 0.6 28
DIDS 1 0.5 23
z
w DIDS 10 0.1 5

0
Oubain 100 0.6 23
w
(I)

DISCUSSION

TIME (minutes) The delay in red cell suspension sedimentation suggests

either aggregation (ie, red cells will not settle until aggre-
Fig 1. Sedimentation as a function of time at different Hct
gates of sufficient size are formed) or cooperatively (ie, the
values. 0. Hct of 26; , Hct of 28; i, Hct of 34; A, Hct of 45.
rate constant for the sedimentation increases with time). Red

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cell sedimentation kinetics is not compatible with either.
zero (phase I of the curve). The next segment (phase 2) is Simple aggregation would require the sedimentation rate to
linear: sedimentation continues at a constant rate. Finally, in increase with an increasing concentration of erythrocytes
phase 3 the rate levels off to zero: sedimentation is com- (Hct); in fact, the opposite occurs. As shown in the Appen-
pleted. dix, the equation describing sedimentation kinetics does not
At different red cell concentrations (Hct range, 10 to 50) fulfill the criteria for cooperativity.
the shape of the curve is qualitatively the same; however, Aggregation of red cells to form uniform spheres. The
there is a shift to the right as the Hct increases (Fig 1): the mechanism of rouleau formation has been extensively inves-
delay (phase 1 ) increases and the slope in phase 2 decreases. tigated.’4 Our data allow further elaboration of the mecha-
This results in slower sedimentation at a gher Hct value. nism of aggregation.
Effect of membrane transport inhibitors on sedimenta- Red cells repel each other because of their negative
(ion. DIDS, an irreversible inhibitor bf anion transport, surface charge from sialic acid residues. At the same time
inhibits both rouleau formation and sedimentation in a they are attracted by the van der Waals forces, which are
dose-dependent manner. Ouabain (0.1 mmol/L) has no electrodynamic in nature. The balance of these two forces
effect on the sedimentation rate. Table 2 shows the slope of determines the most stable arrangement of red cells in an
the sedimentation curve and the ESR as a function of electrolyte solution in the absence of other forces. Assuming
inhibitor concentration. a discoid shape for red cells, van Oss and Absolom’ calcu-
Direct observation of aggregation during sedimenta- lated that at an intercellular distance of 103 nm there is a
(ion. In phase I the homogeneous red cell suspension forms minimum of -4.6 x I02 ergs/cell in the potential energy
a three-dimensional network (Fig 2) that, late in this phase, distribution of erythrocytes suspended in isotonic saline. The
rearranges to form spheres of equal size. This is a sudden distance of the closest approach for erythrocytes increases
change. It can be observed with the naked eye in the with decreasing electrolyte concentration because the repul-
Westergren sedimentation tube: the homogeneous cell sus- sive force ( potential) increases with decreasing ionic
pension suddenly becomes granular in appearance, with the strength and the van der Waals forces are unchanged. (This
spheres visible at the sides of the tube. In phase 2 these explains why there is no rouleau formation in the absence of
spheres sediment at a steady rate (Fig 3). Figure 4 is a electrolytes, eg, in sucrose solutions.) Therefore, in the
photomicrograph of the interface between the sedimented presence of electrolytes, the most stable configuration of
cells and the plasma. The sedimenting entities are the erythrocyte suspensions is a chain of cells attached by their
spheres formed from the rouleaux. The extent of aggregation flat surfaces. However, this energy is small relative to the
does not change during phase 2. To demonstrate that the red energy from Brownian motion (kT); therefore, cells would
cells form fairly stable aggregates during the steady phase of align only transiently but would not form stable structures.
sedimentation (ie, after the delay period) the ESR tube was For this reason red cells will not form rouleaux if the
gently inverted (ie, turned upside down). Sedimentation suspending solution contains electrolytes only.
continued at the same steady rate without a second delay For rouleau formation to occur, macromolecules must be
period. present in the suspending medium.’6 Chien and Jan’7 showed

Table 1 . Measured and Derived Para meters Describing the Curves in Fig 1

Hct 5o a, 2 (1 00 x mm/sI Delay (mm)

25.8 152±2 2,645±56 11±2 2.4 6.5

28.6 152 ± 2 3,081 ± 82 29 ± 3 2.1 8.0
35.8 123 ± 4 4,629 ± 239 31 ± 9 1.6 13.0
44.7 80 ± 10 26.324 ± 1,545 -38 ± 35 0.5 30.0
1574 THOMAS L. FABRY

Fig 2. Photomicrograph (original magnification x40; current

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magnification x 1 6) of sedimenting erythrocytes in a 0.2-mm-
diameter tube. The rouleaux are starting to form spheres: phase 1
ofFig 1.

that increasing the length of a macromolecule (dextran) will

increase the intercellular spacing of the red cells in the
Fig 4. Photomicrograph (original magnification x 40; current
rouleau. He concluded that the macromolecule actually
magnification x 1 6) of sedimenting erythrocytes in a 0.2-mm-
bridges two erythrocytes and only elongated macromolecules diameter tube at the interface between the clear plasma and the
of sufficient length can serve this function. For example, sedimented red cells: phase 3 of Fig 1 . Note that the sedimenting
fibrinogen but not albumin meets the shape requirements to entities are spheres formed from rouleaux and not individual red
cross-link erythrocytes. Therefore, sedimentation will not cells.

take place in serum. It has not been made clear until now,
sites (band 3 protein)’9 per erythrocyte and about I0
however, that the macromolecule has to be either neutral or
fibrinogen molecules are bound to each cell at equilibrium.20
negatively charged (as opposed to positively charged).
Because fibrinogen binding is weak, the 100:1 ratio of
The receptor (binding)site for the macromolecule on the
binding sites to bound molecules is the right order of magni-
erythrocyte membrane has not been identified. We have
tude. In the case of rouleau formation, the two ends of the
shown that DIDS, an inhibitor of anion transport, interferes
macromolecule bind to adjoining erythrocytes to form the
with formation of the aggregates in either plasma or dextran.
aggregates.
DIDS at similar concentrations inhibits the chloride-bicar-
In contrast to the aforementioned mechanism, positively
bonate exchange across the red cell membrane.’5 This sug-
charged macromolecules are not able to bridge the erythro-
gests that the macromolecule binds at the anion transport
cytes in this fashion; they react with the sialic acid residues
site. DIDS also reacts with lysine residues on the membrane
and form amorphous aggregates.2’ This process is not inhib-
surface. If the fibrinogen binding involves these lysine resi-
ited by DIDS.
dues, then DIDS would interfere at this point. Against this
After the formation of the rouleaux, the chains form
latter possibility is the fact that there are 106 anion transport
spheres ofuniform size (Fig 3). The driving force may be the
reduction of the surface free energy. Uniform size is proba-
bly the result of competition between the attractive forces
between cells and the hydrodynamic forces that tend to
reduce the size of the spheres.
The spheres contain not only erythrocytes but also the
macromolecules binding them together. This is in agreement
with the findings of Janzen et a122 who demonstrated binding
of fibrinogen to red cell surfaces. It also explains why
increased Hct values do not increase the rate of sedimenta-
tion: the formation of the spherical aggregates is limited by
the availability of fibrinogen or other plasma macromole-
cules linking the erythrocytes.
In summary, the mechanism of red cell aggregation to
spheres of uniform size proceeds via the following steps: (a)
electrostatic forces draw erythrocytes together to about 100
Fig 3. Photomicrograph (original magnification x 40; current
magnification x 1 6) of sedimenting erythrocytes in a 0.2-mm-
nm, with the discoid shapes lining up parallel to one another.
diameter tube: phase 2 of Fig 1 . Note the uniform size of the This requires the presence of electrolytes. (b) If a neutral or
spheres. negatively charged macromolecule of length greater than
ERYTHROCYTE AGGREGATION AND SEDIMENTATION 1575

100 nm is present, it will bind to the anion transport sites of Table 3. Hct Correction of Sedim entation by Usi ng Equation 1
adjoining erythrocytes. The energy of this binding will Sed Sad ESR
stabilize the parallel configuration of red cells, and rouleaux Hct (observed) ESR (Hct - 40) (Hct - 40)

are formed. (c) Rouleaux rearrange to form spheres, which 25.8 80 80 41 41

reduces the surface free energy. (d) The kinetics of sedimen- 28.6 70 65 40 37
tation must be described by at least two independent parame- 35.8 46 42 38 33
ters (eg, delay before sedimentation starts and steady-state 44.7 22 11 36 18

rate of sedimentation). For a mathematical analysis see the Sed (observed) and ESR are experimentally determined (see the text).
Appendix. Sad (hct - 40) and ESR (Hct - 40) are the calculated corrected values.
Clinical correlation: correctionfor Hct. In clinical prac-
tice there are two reasons for variations in the sedimentation
APPENDIX
rate: (a) its dependence on Hct and (b) its dependence on the
plasma protein concentration. The latter makes it useful as By using SAS for modeling, the best functional form of the
an empirical index of disease activity. In Fig 5 the radius of kinetics of erythrocyte sedimentation is as follows: sedimentation -

the sedimenting sphere (which is directly proportional to the a0 x t2/(a, + a2 x t + t2) (equation 2), where t is the sedimentation

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time and the a’s are experimental parameters. For a cooperative
true rate of sedimentation) is shown as a function of Hct and
phenomenon a2 - 0.
plasma protein concentration. The radius is increased by
Two additional descriptive parameters can be derived from the
either the Hct or increasing
decreasing the plasma protein
three parameters in equation 1 . The first is the delay before
concentration. Therefore, a change in Hct masks the effect of sedimentation starts, ie, the length of phase I . The second is the slope
changing plasma protein concentration and composition. of the linear part of phase 2. These two entities are related to the
Because the steady-state sedimentation (slope in phase 2) in parameters in equation 2: the slope is the value of the first derivative
the range of Hct 10 to 50 is a linear function of Hct (Fig 6), of equation 2 when the second derivative is equal to zero and the
correction can be made by interpolating to a set value, eg, to delay is the x-intercept of the straight line originating from the
an Hct of 40. This eliminates the variations in the sedimenta- inflection point of this curve and having the calculated slope.
tion rate due to the changes in Hct. In practice a good Alternatively, these can be directly measured from the plot of the
curves in Fig I . Table 1 gives the values for these parameters for the
estimation of the steady-state sedimentation can be obtained
curves from Fig 1.
by measuring the amount of sedimentation at 30 and 60
The linear part (phase 2) of the sedimentation results as the
minutes and multiplying their difference by 2 (this way the
spheres formed from the rouleaux fall in a viscous medium. The
sedimentation
Because
55, one
the
can
will have a value that is similar
value of the slope approximates
obtain
to the ESR).
0 when Hct =
the value at an Hct of 40 from the value
radius R of this sphere can be calculated
equation: R2 = 9 V /2(a2 _ from the Einstein-Stokes
,)g (equation 3), where ii is the plasma
viscosity, a, is the plasma density, and o is the red cell density. The
measured at any other Hct by the relationship: Sed (Hct = densities and viscosities are literature23 values, and v is the measured
40) = Sed (observed) x (55 - 40)/(55 - Hct), (equation rate of sedimentation in the linear phase (phase 2).
I ), where Sed (observed) is the sedimentation observed at the These radii can also be measured in the photomicrographs. Figure
6 shows both the calculated and measured values for the radii as a
given Hct and Sed (Hct = 40) is its value corrected to Hct
40. function of Hct for seven samples for a series of different Hct values.
Because the concentration of the bridging macromolecules was
Table 3 shows both Sed (observed) and ESR and their
different in each series, the radius is expressed relative to that at Hct
values corrected to Hct 40 by using equation I . In a larger
35. There is an excellent agreement between calculated and mea-
series of patients with inflammatory bowel disease we sured values.
obtained identical results. Clearly Sed (Hct = 40) provides
an index of disease activity that is fairly independent of Hct. ACKNOWLEDGMENT

Valuable discussions with Drs iean-Claude iustice and Marie-

Claude Justice and the assistance of Dr Steven Dikman with the
‘NN,AO I I I I
2.0 photomicrographs are gratefully acknowledged. I am very grateful
U) to Karen Sadock for her editorial review.
0 .6
a:
. #{163}:4NA

0.4- , , I

0 0 20 30 40 50
H EMATOCRIT

Fig 5. Radius of the spherical aggregates at different Hct

values. Erythrocytes are suspended in autologous plasma and Fig 6. Radius of spherical aggregates as a function of Hct. The
serum mixtures. The third coordinate is the fraction of plasma in seven different symbols represent different blood samples. The
the suspending medium. radius is expressed relative to that observed at Hct 35.
1576 THOMAS L. FABRY

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