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Reticulocyte Rigidity and Passage Through Endothelial-like Pores
By Richard E. Waugh
The importance of cell rigidity in regulating the release of
reticulocytes from the bone marrow has been investigated in
a model system. Reticulocytes were obtained from phlebotomized rabbits and separated from whole blood by discontinuous density gradient centrifugation. The mechanical properties of the cells were tested. Using single-cell micromechanical
techniques, the membrane elastic rigidity and the viscoelastic response of reticulocyte and mature cell populations were
measured. The reticulocyte membranes were more rigid than
the mature membranes, but the reticulocyte properties were
heterogeneous, and some cells exhibited behavior indistinguishable from the mature cells. The mean time constant for
viscoelastic recovery was the same for reticulocytes as for
mature cells, but the variability within the reticulocyte population was greater. The possible influence of this increased
rigidity on cell egress from the bone marrow was tested
using an in vitro model of the thin endothelial pores found
within the marrow. A silicon wafer approximately 0.1 p m in
thickness and containing a small (1.2-pm diameter) pore in its
center was cemented over the tip of a large (15.0-pm ID)
micropipette. The passage of cells through the pore was
observed as a function of the pressure across the pore.
Consistent with the difference in mechanical properties, the
reticulocytes required greater pressures (as great as 4.0 mm
Hg compared with < 1.O mm Hg) and took longer t o traverse
the pore. These measurements support the postulate that
deformability is important in the regulation of the release of
cells from bone marrow.
0 1991by The American Society of Hematology.
B
g~adient.4.’~
Such a mechanism would be consistent with the
postulate that the passive deformability of the cells is a
regulator of egress, but the origin and magnitude of such a
pressure gradient is not known.
In previous reports, we have examined the postulate that
egress is pressure-driven, and have developed a theoretical
model for predicting the magnitude of the driving pressure
and the time that it would take a cell to complete passage
through a small circular a p e r t ~ r e .We
’ ~ tested these theoretical predictions in a model system in vitro using a thin
silicon wafer with a small (1.0 to 2.0 p,m) diameter pore in
its center to mimic the geometry of the cellular deformations in the marrow.I4We demonstrated that the pressure
needed to drive a mature erythrocyte through such a pore
was 1.0 to 3.0 mm Hg, well within the physiological range,
and that the time for passage was on the order of 1 second.
In the present report, we examine the mechanical properties and passage time of stressed reticulocytes from the
rabbit, and discuss the implications of our observations on
postulated mechanisms of controlling erythroid egress in
the marrow.
ONE MARROW contains two topologically distinct
spaces: the hematopoietic space where cells grow and
differentiate, and the vascular bed, which is contiguous with
the rest of the circulation.’.*These two spaces are separated
by a thin layer of adventitial and endothelial cells. To enter
the circulation, cells must pass through small pores in the
thin endothelial
To maintain homeostasis in a
normal adult human, approximately 2.5 million new red
blood cells must pass into the circulation each second. In
the case of some illnesses or blood loss, the demand for new
cells is even greater. Despite the importance of marrow
egress and its control, important questions remain about
both the mechanism that powers the passage of cells across
the endothelium and the processes that control the cell flux.
Two important factors that are likely to be involved in
controlling cell egress have been recognized. Early investigators demonstrated that the deformability of erythroid
cells increased as the cells matured.6These observations led
to the postulate that the passage of cells into the blood
stream might be limited by cell deformability. Subsequently, adhesion of immature cells to extracellular matrix
components within the marrow was also implicated as a
possible mechanism for controlling cell egress.’ It is known
that a number of surface markers appear and disappear
during erythroid maturation,’-’’ and it has been demonstrated in culture that the affinity of maturing erythroleukemic cells for fibronectin decreases with maturation.” These
observations led to speculation that the less mature cells
may not have access to the endothelial pores because of
their adherence to extracellular matrix components in
other regions of the hematopoietic compartment. In the
living marrow, it is likely that both cell adhesion and cell
deformability participate in the control of cell egress.
The driving force for red blood cell egress is even less
well understood. Clearly, the motility of leukocytes makes it
possible for these cells to leave the marrow under their own
power. Cytokinesis has been observed in marrow reticulocytes,” but the movements of reticulocytes are small and
slow compared with those of leukocytes, and directed
motions of these cells have not been demonstrated. Observations of the shape of reticulocytes during egress led us to
postulate that the process may be driven by a pressure
Blood, Vol78, No 11 (December l ) , 1991: pp 3037-3042
MATERIALS AND METHODS
Cell preparations. Reticulocytes were separated from whole
blood by discontinuous density gradient centrifugation.” To obtain
From the Department of Biophysics, University of Rochester, School
of Medicine and Dentisoy, Rochester, NY.
Submitted August 20, 1990; accepted July 24, 1991.
Supported by the Public Health Service under National Institutes of
Health Grant No. HL 18208. The work was performed in part at the
National Nanofabrication Facility which is supported by the National
Science Foundation under Grant No. ECS-8619049, Come11 University, and industrial affiliates.
Address reprint requests to Richard E. Waugh, PhD, Department of
Biophysics, University of Rochester, School of Medicine and Dentistry,
601 Elmwood Ave, Rochester, NY 14642.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with I8 U.S.C. section 1734 solely to
indicate this fact.
0 1991 by The American Society of Hematology.
0006-4971191 I781 I -0010$3.00l0
3037
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3038
a sufficient fraction of reticulocytes in the circulation, the rabbit
was bled at a rate of 8 mL/kg body weight on the sixth and third
days before the experiment. This resulted in a circulating reticulocyte fraction of 10% to 15%. On the day of the experiment, a
sample was drawn in EDTA, the cells were washed in phosphatebuffered saline (PBS) (134 mmol/L NaC1, 5.0 mmol/L KCI, 1.4
mmol/L NaH,PO,, 8.6 mmol/L Na,HPO,, pH 7.3 to 7.4) plus 1.0
mmol/L glucose, and layered onto discontinuous density gradients made using dilutions of arabinogalactan. The densities of the
layers were approximately 1.220 g/mL, 1.110 g/mL, 1.100 g/mL,
1.088 g/mL, 1.077 g/mL, and 1.066 g/mL. After centrifugation at
27,OOOg for 90 minutes, the cells were harvested from the gradients
in three groups. The top fraction contained most of the reticulocytes, and the middle and bottom fractions each accounted for
roughly half of the remaining cells. Each group was washed five
times in PBS plus 1.0 mmol/L glucose. After washing, the fraction
of reticulocytes in each fraction was determined by staining with
new methylene blue, and a sample of each group was suspended in
PBS plus 4.0% acid-citrate-dextrose plasma for measurement.
In early experiments, arabinogalactan (Stractan) was obtained
from Sigma (St Louis, MO), and desalinated in our laboratory
according to the method of Clark et a].'' Raw powder was mixed
with anion exchange resin and allowed to stand for 30 minutes at
0°C. The resin was separated from the Stractan by filtration. The
process was repeated with a cation exchange resin. The purity of
the preparation was assessed by measuring the osmolarity of the
solution by vapor pressure osmometry. A value less than 100
mOsm/kg was considered acceptable. In later experiments, purified arabinogalactan (Larex-L.O.) was obtained from Larex International (Tacoma, WA) and used directly.
Surface rigidity. The procedure for measuring membrane deformability has been described in detail elsewhere.I6 Briefly,
micropipettes with inside diameters of approximately 1.0 to 1.2 pm
were made by fracturing off the tips of capillary tubing pulled to a
needle point on a vertical pipette puller (David Kopf Instruments,
Tajung, CA). Pipettes were filled with filtered and degassed PBS
and inserted into the cell suspension through the open side of a
U-shaped cavity between two cover glasses on the microscope
stage. The pressure at the tip of the pipette was controlled by
adjusting the position of a water-filled reservoir connected to the
back of the pipette. Zero pressure was determined by observing the
movement of cells or small particles at the pipette tip, and suction
pressures were applied by adjusting the level of the reservoir
relative to the zero point using a micrometer drive. The accuracy of
the zero pressure was plus or minus 0.5 mm H,O and the accuracy
of changes in pressure relative to the zero was less than 0.1 mm
H,O. A cell was aspirated at an initial pressure of approximately
2.0 mm H,O in a relatively flat region of the surface, and then the
aspiration pressure was increased stepwise in increments of 1.0 to
2.0 mm H,O. Experiments were recorded on videotape and the
length of the cell projection into the pipette was measured as a
function of the aspiration pressure from the recorded images. The
surface rigidity was characterized in terms of the membrane shear
modulus, k, which is related to the slope of the length-pressure
data pairs (dL/dP)I6:
where R, is the pipette radius.
Surface viscosity. The time constant for cell recovery was
determined using the whole cell recovery technique developed by
Hochmuth et a1.17Cellswere allowed to settle to the glass surface at
the bottom of the chamber and adhere. A micropipette was used to
free cells from the surface except at a single point, and then the
RICHARD E. WAUGH
cells were extended between the pipette and the attachment site on
the glass surface by slowly withdrawing the pipette. After release,
the cell recovered rapidly to its initial geometry. The recovery was
recorded on videotape and the time course of the recovery was
determined by frame-by-frame playback of the recording. Temporal resolution was limited by the video framing speed (%o s). The
time constant t, for the recovery was determined by least squares
regression to a prediction for the recovery of a rectangular strip of
membrane:
x
x-
+ e-lllc
L/W
=
(LIW), .
~
e-t/4
'
where
and where L/W is the ratio of the cell length to the cell width,
subscript o indicates the resting state and subscript e indicates the
initial extended state Gust after release). According to the Kelvin
model for cell viscoelasticity, the time constant t, is the ratio of the
membrane viscosity to the membrane elastic shear modulus."
Pore passage. Pipette pores were made by cementing silicon
wafers over the tip of a large (15 pm) mi~ropipette.'~
The wafers
were manufactured at the National Nanofabrication Facility (Cornell University, Ithaca, NY) using wafer fabrication techniques.
The wafers consisted of a thin membrane of silicon-nitride with a
perforated ring etched through it and a circular pore ( - 1.0-pm
diameter) etched through the center of the disk formed by the
perforation. A microforge was used to fracture off the tip of a
capillary needle to form the pipette, and the resulting pipette was
bent to a 90" angle to facilitate assembly. A second, smaller pipette
was used to paint a ring of ultraviolet-curing adhesive (Crystal
Clear, Loctite, Cleveland, OH), and the larger pipette was pressed
onto the adhesive to form a seal between the wafer and the pipette.
After exposing the adhesive to high-intensity microscope illumination, the pipette could be withdrawn, breaking the pore away from
the wafer along the perforated ring. The assembled pipette pore
was filled with PBS and inserted into the cell suspension in a small
chamber on the microscope stage. The pressure control system was
the same as that used in the membrane rigidity measurements,
except that additional changes in pressure could be introduced by
evacuating the air space above the water reservoir with a syringe.
Another pipette was used to position a cell opposite the pore while
the pressure was set to an appropriate value. Then the cell was
released and allowed to be drawn to and traverse the pore (Fig 1).
The passage was recorded on videotape, and the recordings were
used to measure the time of passage as a function of the pressure
across the pore.
RESULTS
Because it is not always possible to distinguish reticulocytes from mature cells by direct visual observation, it was
essential t o obtain relatively pure samples of reticulocytes
and to assess the reticulocyte fraction by staining. The
samples we obtained using the density fractionation method
described above resulted in a cell population that was 90%
to 95% reticulocytes. Many of the reticulocytes were
biconcave in shape, but other more complicated shapes
were also evident. The biconcave cells often contained
reticular material that was visible in the microscope under
monochromatic (436 nm) brightfield illumination.
Cell rigidity. The reticulocyte population exhibited
greater heterogeneity and tended to be more rigid than the
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3039
RETICULOCYTE RIGIDITY AND EGRESS
6.07
4.0-
a
p:
3
2.0-
0.0 1
0.00
I
I
I
1
0.01
0.02
0.03
0.04
P x Rp (dyn/cm)
Fig 2. The length of the cell projection (divided by the pipette
radius) as a function of the pipette aspiration pressure (times the
pipette radius). The shaded region shows the distribution of measurements for the mature rabbk cells. The data points represent individual
measurements on a reticulocyte, each different symbol representing
data from a different cell. Solid lines are the linear regressions to the
data for individualcells. The reticulocytes showed greater heterogeneity in behavior than mature cells and typically required larger pressures to form projections.
B
Fig 1. Photographs from the television monitor showing the
passage of a reticulocytethrough a pore pipette. (A) Before release, a
small pipette is used to position the cell opposite the pore while the
pressure across the pore is adjusted to the proper value. (6) After the
pressure is adjusted, the cell is released from the small pipette and
enters the large pipette through the pore. (Bar = 10 pm.)
denser, more mature fractions of cells. This is evident from
the shorter projection lengths that were obtained for the
pressures applied (Fig 2). Two features of the length
pressure data are noteworthy, namely, the initial projection
length tended to be shorter for the reticulocytes than for
the mature cells, and the slope of the length-pressure
curves is shallower, ie, the increase in projection length for
a given increment in pressure was smaller. The decrease in
the initial projection was proportionately greater than the
decrease in slope, so the data for the reticulocytes are
displaced vertically on the length-pressure graphs. Statistically, the difference between the slopes of the lengthpressure curves was barely significant (f = .03, SmithSatterthwaite, modified Student's t test), but the difference
in the y-intercepts for the curves (L/&) was highly
significant (P= .001, Table 1).
Recovery time constant. The reticulocytepopulation also
exhibited greater heterogeneity in the rate at which the
cells recovered their resting geometry after extension (Table 2). However, there was no difference in the mean value
for the recovery time constant between the reticulocytes
and the "middle" cell fraction, although the densest frac-
tion recovered more slowly than the other two. The observation that dense cells recover more slowly than less dense
cells confirms observations made previously by other invest igators.'9.?0
Passage through pores. The increased heterogeneity and
increased rigidity of the reticulocyte populationis reflected
in their ability to traverse thin small pores similar to those
found in the bone marrow. There was a general trend
toward greater pressures required for passage to occur, and
longer passage times. However, as in the case for cell
rigidity, there was a great deal of heterogeneity within the
reticulocyte population. Although some cells were significantly more rigid than mature cells, others exhibited behavior indistinguishable from the mature population (Fig 3).
The potential for deformability to be a discriminatory
factor in cell release is illustrated in Fig 4. The fraction of
the cells tested that could complete egress is shown as a
function of the pressure across the pore. For this sized pore,
all of the cells in the mature population would pass through
the pore at a pressure of 2.0 mm Hg, but only 25% of the
reticulocyteswould pass at this pressure.
Table 1. Rigidity Coefficients of Reticulocytes and
Density-FractionatedCells
u (dynelcm)
LIR,
Fraction
(SD)
(SD)
n
Top (reticulocytes)
0.00460*
1.40"
(0.42)"
1.74"
(0.26)
2.12"
(0.25)
24
Middle
Bottom
(0.00142)"
0.00385
(0.00044)
0.00398
(0.00053)
"Significantly different from all other samples (P < .05).
10
22
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3040
RICHARD E. WAUGH
1 .oq
Table 2. Recovery Time Constant for Reticulocytesand
Density-Fractionated Cells
Fraction
t(S)
SD
n
Top (reticulocytes)
Middle
Bottom
0.131
0.131
0.149*
0.040t
0.025
0.031
40
15
24
-c
*Significantly different from all other samples (P < .05).
tSignificantly different from middle fraction (P < .05).
Reticulocyte heterogeneity. A separate series of measurements were performed to examine the variability in properties within the reticulocyte population in greater detail.
Whereas in the experiments described above, reticulocytes
with discoid shapes were selected so that comparisons with
mature cells would not be complicated by shape differences, in this series, cells with a variety of shapes were
selected. These were subjectively classified as lobular,
intermediate, or discoid. (Reticular material was observable in all of the discoid cells.) Twelve different reticulocytes were tested. On each cell, measurements were made
of both surface rigidity and passage time. Two important
observations were made. First, the lobular cells were
consistently more rigid than most discoid cells. This is
illustrated in Fig 5A, which shows the length of the
membrane projection in a micropipette as a function of the
aspiration pressure for three lobular and three discoid cells.
Second, we observed that the cells with greater membrane
rigidity exhibited larger threshold pressures and slower
transit times at a given pressure during passage through a
silicon pore. This is illustrated in Fig 5B,which shows the
passage time as a function of pressure for the same six cells
from which the data in Fig 5Awere obtained.
DISCUSSION
Increased rigidity of reticulocytes. These results confirm
previous studies that have shown an increased rigidity of
35.0
30.0
25.0
h
23
:.
20.0
15.0
i-
10.0
5.0
0:o
2:o
4:O
6:O
8:0
Pressure (torr)
Fig 3. The time required to complete passage through a pore 1.2
pm in diameter as a function of the pressure difference across the
pore. The shaded area shows the distributionof times for the mature
cell population, and the data points represent individual measurements on a reticulocyte, each different symbol representing a different cell. As in the micropipette measurements, the reticulocytes
exhibited greater heterogeneitythan the mature cells, and generally
required larger pressuresto complete passagethrough the pore.
E
Reticulocytes
0.0
0:o
2.0
4:O
6.0
Pressure (torr)
Fig 4. The fraction of cells passingthrough the pore in less than 1.0
minutes as a function of the pressure difference across the pore. 0 ,
Results for the mature cells (n = 10); W, results for the reticulocytes
(n = 16). These results illustrate the potential for discriminatory
release of more deformable cells from the marrow.
immature erythrocytes. Leblonde et aI6 examined the deformability of marrow reticulocytes and normoblasts from
mouse and humans and observed a progressive increase in
cellular deformability with increasing cell maturity. More
recently, Chassis et all2 have examined the deformability
and stability of reticulocytes from rat bone marrow and
from patients with elevated circulating reticulocyte fractions (10% to 15%). They observed increasing deformability and membrane stability with increasing cellular maturity
(as assessed morphologically). Interestingly, they could not
detect differences in rigidity (using the ektacytometer)
between late-stage reticulocytes (those having a biconcave
shape) and mature cells. Our results contrast with theirs
somewhat in that even among the biconcave reticulocytes,
we found cells with significantly greater rigidity than the
mature cells. However, when comparing cells within the
reticulocyte population, we too found that the cells with a
shape characteristic of a less mature cell were consistently
more rigid than cells with a more discoid shape.
The appearance of cells with elevated rigidity in the
circulation (that we observed in the present study) may not
occur under normal conditions, but may be the result of the
stressed conditions under which the cells were obtained.’ It
is generally thought (although it has never been demonstrated) that these immature cells will continue to mature
after release from the marrow and will eventually attain
normal deformability. However, there is preliminary evidence that the increased rigidity of prematurely released
cells may persist under some conditions. Recently, Meier et
a]*’ measured the deformability of red blood cells from
uremic patients before and after treatment with erythropoietin, Before treatment, the patients were anemic and their
red blood cells exhibited increased membrane rigidity as
assessed by micropipette aspiration. After treatment, not
only did hematocrit return toward normal, but the increase
in membrane rigidity was also alleviated. This result is
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3041
RETICULOCYTE RIGIDITY AND EGRESS
4.01
A
I
0.0
0.00
= f
I
I
I
I
I
0.01
0.02
0.03
0.04
0.05
P x Rp (dyn/cm)
'"01
n
2
15.0
4
10.0
\v
5.0
B
0.0
0.0
0:o
1 .o
2.0
3.0
4.0
Pressure (torr)
Fig 5. (A) Length of the cell projection (divided by the pipette
radius) as a function of the pipette aspiration pressure (times the
pipette radius). Open symbols represent data for discoid cells. Solid
symbols represent data for lobular cells. Lobular cells consistently
exhibited greater rigidity than discoid cells. (B) Time required to
complete passage through a pore approximately 1.4 pm in diameter
as a function of the pressure difference across the pore. Data were
obtained from the same six cells from which the data in (A) were
obtained, and symbols of the same type correspond to data obtained
on the same cell. Cells with greater rigidity exhibited larger threshold
pressures and took longer to traverse the pore at a given pressure.
Note that the cell representedby the solid circles did not traverse the
pore at pressures less than 1.5 mm Hg.
surprising, because the uremic conditions originally thought
to account for the increased rigidity (by oxidative damage)
are not altered by the treatment. One possible explanation
for these observations is that because of stressed (anemic)
conditions, cells with increased rigidity were released into
the circulation, and this increased rigidity persisted for the
life of the cell. Late-stage maturation of reticulocytes is
thought to occur in the ~ p l e e nRecently,
.~
Noble et aIz2have
shown that sequestration of reticulocytes in the spleen is
reduced under stressed conditions. Thus, under conditions
of chronic anemia and in the absence of mitigating factors,
an increase in the rigidity of the circulating cell fraction
might be expected.
Implications about the control of cell egress. Mechanical
analysis of the passage of cells through poresL3enables us to
evaluate some postulates about the control of egress. Two
aspects of the analytical results are relevant. First, the
minimum pressure required for egress (threshold pressure)
is a strong function of the pore radius, increasing as the
pore radius decreases. Second, the threshold pressure
increases with the elastic rigidity of the cell membrane (see
Fig 5). The dependence of the threshold pressure on pore
radius is exemplified by the fact that the threshold pressure
observed for mature cells in the present study (R, = 0.6
Fm) is approximately two times larger than that observed in
the previous study (RP = 0.8 pm).I4 Because the threshold
pressure is directly proportional to the surface rigidity (to a
first approximation), the difference in the minimum pressure between reticulocytes and mature cells in the present
study is approximately twice as large as it would have been
for the larger pore used in the previous study. For example,
consider a mature cell with a threshold pressure of 1.0 mm
Hg f0r.a 1.2-pm diameter pore and a reticulocyte with a
threshold pressure of 4.0 mm Hg. The difference between
the threshold pressures (3.0 mm Hg) is a significant fraction
of the total pressure measured between the marrow space
and the emissary vein just outside the cortical bone in
rabbits (-20 mm Hg).23If the pore had a diameter of 1.6
pm, the threshold pressures would be reduced by half to 0.5
and 2.0 mm Hg, respectively. In this case, the difference is
only 1.5 mm Hg, which may not be sufficient to select in
favor of the more deformable cell. Conversely, the analysis
predicts that if the pore diameter were reduced to 0.9 pm,
the threshold pressure should double, making the difference in threshold pressure between the reticulocyte and the
mature cell larger, approximately 6.0 mm Hg. Thus, the
smaller the pore, the greater the ability to discriminate
between cells of different deformabilities. Clearly, changing
the pore radius would be an extremely effective way of
changing the type of cell (more or less deformable) that is
released from the marrow.
Much remains to be learned about the mechanism for
controlling the flux of new red blood cells from the bone
marrow. Although adhesion to fibronectin may play a role
in retaining immature cells in the marrow,' the observation
that in splenectomized animals a large fraction ( 50%) of
the circulating cells are adherent to fibronectid argues
against this as being the exclusive mechanism for retaining
immature cells in the marrow. The results of the present
study clearly indicate the potential to distinguish between
mature and immature cells on the basis of their deformability. Previously, on the basis of an analysis of the flux of cells
across a porous interface, we argued that changes in the
pore dimensions would be one of the most effective ways of
controlling the flux of cells from the m a r r ~ w . ~Both
. ' ~ the
flux of cells across the endothelium and the ability to
distinguish between cells of different deformability are
strong functions of the radius of the endothelial pores. As
the radius of the pore increases, the flux of cells across the
endothelium increases, but the ability to discriminate among
cells on the basis of their deformability decreases. We
predict that as the demand for cells increases, the mean
-
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RICHARD E. WAUGH
3042
radius of the endothelial pores will increase, leading to the
release of increasingly less deformable (immature) cells.
Thus, our analysis is consistent with the general observation
that the number of immature cells in the circulation
increases under conditions in which the demand for new
cells is elevated? Because of the relative inaccessibility of
the marrow space, it is difficult to test postulated mechanisms for controlling egress. However, measurements of the
size and number density of pores in the marrow under
normal and stress conditions should provide additional
insights into these mechanisms and would allow more
precise estimation of the hydrostatic pressures that would
be needed to drive cells from the marrow.
ACKNOWLEDGMENT
The author acknowledges the technical support of Richard
Bauserman, who constructed the pipette pores and assisted with
the micropipette experiments, and Donna Brooks, who performed
the cell separations and data analysis. He also thanks Dr Margaret
Clark for her advice and assistance in setting up the cell separation
protocols, and Rick Bojko of the National Nanofabrication Facility, who produced the silicon wafer pores.
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1991 78: 3037-3042
Reticulocyte rigidity and passage through endothelial-like pores
RE Waugh
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