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Erythrocyte
Deformation
in Shear
Membrane
Stiffness,
By Kazunori
The
effect
of
viscosity
shear
suspension
using
cells,
hemolyzed
and
external
$
membrane
T IS WELL
viscosity
in a shear
rate,
reduces
At
high
that
shear
The
blood
by
enhanced
and/or
ability
with
Cox,6
by the
in viscosity
increase
in shear rate.
of deformation
lular
viscosity,
respectively.
of suspen-
as well
with
an
against
and
about
flow
and
cell
elongate,
the cell interis frequently
we
stress,
the
stiffness
assessed
quantitatively,
influence
of intracellular
on cellular
In addition
cal phenomena
should
have
deformation.
as an
Rheoscopy”
directly
and
allows
in a diluted
suspension.
cells
is difficult,
however,
for
(Ht)
microscopic
tially
on cellular
globin
ency
cells
of cells
This
and
with
medium
tion of
different
various
kinds
intracellular
and
55%);
and
hardened
shear
and
(c)
the
of
intact
PH
flow
cells.
on
external
viscosities,
the
cells;
of
cells
and
In these
studies,
on the
applied
and
Vol 69. No 3 (March).
hemo-
cell
intact
glutaraldehyde-treated,
ellipsoidal
deformation
The
suppression
through
disturbance
by Grune
1987
of
hard-
of intact
cells
deformation
of laminar
& Stratton.
was
probably
flow-lines
around
Inc.
MATERIALS
Preparation
ofPH
Cells
Pink
were
prepared
nary
AND
METHODS
ghosts
experiment,
but
by hypotonic
ellipsoidal
hemolysis
deformation
flow was disturbed.
Therefore,
we prepared
appropriate
of dextran
by the
amount
in a prehimi-
under
uniform
shear
PH cells containing
following
an
procedures.
Freshly drawn heparinized
blood from healthy donors was centrifuged to remove plasma and buffy coat. The packed cells were then
washed
me,
three
times
with
50 mmol/L
palmitoyl
an isotonic
of HEPES,
The
cells
containing
changed
After
centrifuged
was
of
suspended
St Louis)
PH
allowed
packed
low internal
(heavy
of
5 mmol/L
in
isotonic
to stand
(light
were
(the
in the presence
for 5 minutes
LPC-treated
viscosity
cells),
the
at a Ht of45%
to spheroechinocytes
to obtain
having
(I 15 mmoh/L
of L-a-hysophosphatidylchol-
Chemical,
the suspension
viscosity
buffer
of glucose,
were
38 izmol/L
Sigma
from discocytes
ice, it was
HEPES
6 mmol/L
washed
(LPC,
of LPC).
suspended
in 9 mL
of ice-cooled
cells.
Two
PH cells)
prepared
hypotonic
of HEPES,
I mmol/L
of KCI,
of EGTA, 5 mmol/L
of MgSO4,
30
by
on
kinds
and high
incubating
mOsm/kg)
containing
7%
Sweden)
and
Uppsala,
gentle
stirring
HEPES
I mmol/L
0.8 mmol/L
Dextran
was
(designated
T-40
incubated
as step-I
cells
buffer
(Pharmacia
for
(10
of glucose,
2
of ATP; pH
Fine
5 minutes
suspension).
on
In step
2,9
In step
3, to return
the
isotonicity,
1 .44
mL
of a hypertonic
HEPES
buffer
(1,150
mmol/L
of NaCI,
500 mmoh/L
of HEPES,
60
mmol/L
of glucose, 50 mmol/L
of KCI; pH 7.4) was added to the
basis
shear
intact
of
of deformacells
Ht range
having
intact
cells
in
the
cell
two parameters
the
related
in
variable
internal
to membrane
and
ofPhysiology,
Shigenobu.
andfrom
Ehime
reprint
School
©
Ehime,
requests
ofMedicine,
Medical
ofMedicine,
Ehime
Japan.
September
the Ministry
25. 1986.
of Education
of
Foundation.
to Dr
Ehime
Ehime 791-02, Japan.
The publication
costs ofthis
charge payment.
This article
“advertisement”
in accordance
indicate
School
Onsen-gun,
May 21, 1986; accepted
in part by grants
from
Address
ogy,
mixed
deformation
of an empirical
stress,
Japan
4% to
a
the Department
Submitted
Supported
of Ht
(from
From
University,
glutaraldehyde-treated,
T. Shiga,
University.
Department
of Physiol-
Shigenobu,
Onsen-gun.
article were defrayed
in part by page
must therefore
be hereby
marked
with /8 U.S.C.
§1734 solely
to
this fact.
I 987 by Grune
& Stratton,
Inc.
0006-4971/87/6903-0003$3.00/0
stiffness.
Blood,
on
When
transpar-
(b) the influence
a wide
behavior
Ht.
mL of the same hypotonic
7% dextran
solution
was added to the
step-i solution, and the suspension
was stirred gently for 5 minutes.
par-
of viscosity
on the degree
such
as PH
density-fractionated
over
with
effect
a certain
cells.
ice with
of Ht
intracellular
suppressing
viscosity.
rate
intact
disturbed.
7.4,
intensity
prepared
the
mixed
the
Chemicals,
physiological
influence
contribution
of cells,
viscosity,
is discussed
depending
the
shear
deformation
suspension
the
to increase
intracellular
(a)
diamide-treated
on the cellular
we
to study
dextran
maintain
describes:
suspending
cells,
cells)
ie, we replaced
cells
article
of
cells,
mmol/L
mmoh/L
deforma-
in light
Therefore,
(PH
deformation,
of intact
suspension
were
ened
was
of individual
of reduction
cells
intact
The
above
LPC-treated
cells in hypotonic
dextran
solutions as follows.
Light PH cells.
In step 1, 1 mL of the LPC-treated
packed
on cellular
of cell
suspension.
appeared
internal
the cell defor-
degree
observation
the
because
observation.
hemohyzed
us to observe
the
Direct
in
value
factor
cell
deformation
of PH cells,
membrane
to study rheohogirole of hematocrit
influencing
to measure
tion
hematocrit
deformabihity,
blood,
the
diluted
RBCs
of shear
and
Shiga
sion
viscosity
at higher
hematocrits
(Hts)
generally
enhanced
the ellipsoidal
deformation
of cells, in the same
manner as increasing
the suspending
medium viscosity
of a
KCI, pH 7.4).
HEPES
buffer
range
viscosity
Takeshi
of the
intracel-
geometry)
a wide
and
NaCI,
deformation.
to single cell
of circulating
be investigated
mation
over
Hematocrit
medium
been
revealed,7’#{176} the quantitative
contribution
of these
determinants
to deformabihity
is not yet clear.
In the present
study,
Viscosity,
of
that
of liquid
droplet’4
as
ie, the elongation
of cells is
stiffness,
Maeda,
of Internal
as the
flow.’
of suspending
and
S
increase
shear
orient
Influences
occurred
to the
Although
the major determinants
(deformabihity)
of RBCs
(ie,
membrane
t#{149},
and orientation
stably
the analogy
by Taylor5
and
stress,
related
viscosity,
accompanying
membrane
tank-treading
br.2 The cellular
deformation
in shear
treated
described
shear
decreases
RBCs
(DI)
be a function
increase
resistance
stresses,
ie.
intact
index
to
the deformation
hydrodynamic
of cells.
viscosities,
suspension
because
quantitatively
kinds
parameters
properties.
cell
RBC
shown
internal
KNOWN
of red
and
of
is applied
adjustable
viscoelastic
rate
deformation
was
i’ti.,
and
are
shear
hematocrit
various
The
cells
where
Nobuji
density-fractionated
cells.
deformed
a and
cells
cells,
diamide-treated
are
,,,
with
(PH)
)‘
on
and
Kon,
was studied
rheoscope
of ellipsoidally
of
and
and
(depending
medium)
on RBC deformation
a cone-plate
partially
I
force
of extracellular
Flow:
1987:
pp 727-734
727
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KON.
728
step-2
suspension.
isotonic
In step 4, to remove
HEPES
buffer
LPC from cells,
I .5%
containing
bovine
I 8 mL of the
serum
albumin
suspension,
the
Ht
suspension
and
the
was
calculated
MCV
from
oforiginal
the
cell
MAEDA,
number
before
GA
AND
SHIGA
of cells
in the
treatment.
(BSA, fraction-V,
purchased
from Armour
Pharmaceutical,
Kankakee, IL) was added to the step-3 suspension,
followed by incubation
of the suspension
for 1 hour at 37 #{176}C.
During
the incubation,
reseahing of cells was accomplished.
In step 5, the resulting
PH cells
were washed
once with an isotonic
7% dextran
solution
(100
mmol/L
of NaCI, 50 mmol/L
of HEPES,
6 mmol/L
of glucose, 5
mmol/L
of KCI; pH 7.4) to remove a minute amount of membrane
fragment
and washed twice with the isotonic HEPES
buffer.
Heavy PH cells.
The above procedures
for the preparation
of
light PH cells were followed with a minor modification:
In step 2,
instead of the hypotonic
7% dextran
solution,
9 mL of the hypotonic
HEPES buffer containing
30% dextran
was added to the step-i
suspension.
Preparation
ofDensity-Fractionated
Intact
Cells
Electrophoretic
Analysis
The polyacrylamide
dodecyl sulfate (SDS)
metric
scanning
ofMembrane
Proteins
gel electrophoresis
(PAGE)
in 1% sodium
was performed
on a slab gel.’7 The densito-
of Coomassie
brilliant
blue
R-250-stained
gel
was
performed
by a densitometer
(Dual-wavelength
TLC Scanner,
CS900, Shimadzu,
Kyoto, Japan).
After diamide treatment,
the degree
of cross-linking
of membrane
proteins was estimated
by the method
of Maeda and colleagues.’5
Determination
ofAmounts
of Membrane
Lipids
To estimate
the loss of membrane
lipids during the preparation
of
PH cells, the amounts
of cholesterol
and total phosphohipids
were
determined
by the method
of Zlatkis
and Zak’8 and Bartlett,’9
respectively.
Intact
cells were fractionated
by density-gradient
centrifugation.’2 An isotonic suspension
of Percohl (Pharmacia)
in the HEPES
buffer was used: 75.6% Percoll to obtain low-density
cells and 82.8%
Percohl to obtain high-density
cells. One milliliter of packed cells was
poured
on 13.5 mL of Percoll medium
and then spun at 7,000 g for
20 minutes at 20 #{176}C.
The low-density
cells ( 10% to I 5% of all cells)
gathered
in the upper half of the tube (in 75.6% Percoll) while the
high-density
cells (30% of all cells) sedimented
in the lower half of
the tube (in 82.8% Percoll).
The low-density
cells and high-density
cells can be assigned
to young and aged cells, respectively.’3”4
The
fractionated
cells were washed three times with the isotonic HEPES
buffer to remove Percoll. The fractional
percentage
was calculated
on the basis of hemoglobin
concentration.
ent
Preparation
isotonic
ofDiamide-Treated
(Diamide-Treated
The
HEPES
buffer
(diazene
30
minutes
three
times
and
cells were suspended
incubated
with
acid
isotonic
concentrations
bis(N,N-dimethylamide,
at 37 #{176}C
(Ht 10%).”
the
various
in the isotonic
After
HEPES
Preparation
ofGlutaraldehyde-Treated,
Cells (GA-Treated
PH Cells)
of diamSigma)
incubation,
cells
for
were washed
buffer.
Hardened
PH
Light PH cells were suspended
in the isotonic
HEPES
buffer
containing
0.1% glutaraldehyde
at a Ht of 10% and incubated
for 30
minutes at 37 #{176}C
with gentle stirring. To remove unreacted
glutaraldehyde,
cells were washed
three times with the isotonic
HEPES
buffer.
of Cellular
Cellular
deformation
rheoscope1’
constructed
microscope
(Model
0.8
#{176}
flashlight
ofMean
Corpuscular
Volume
(MCV)
MCV was determined
from the number of cells measured
by an
automatic
counter
(Model
CC-i 10, Toa Electric,
Tokyo), and the
packed cell volume was determined
by the method of England
and
Down,’6 as follows. Cells were washed
with
the isotonic
HEPES
buffer containing
I .5% BSA and suspended
in the same solution.
Ten microliters
of ‘3’I-labeled
BSA solution
(150 sCi/mL)
were
added to 5 mL ofthe cell suspension
(Ht 50%). The radioactivities
of
supernatant
and sedimented
fraction were measured
by an autowell
‘V counter
(ARC-605,
Aloka,
Tokyo)
after centrifugation
for 10
minutes at 6,000 g with a microhematocrit
tube using a microhematocrit centrifuge
(Model KH-120,
Kubota,
Tokyo) and the packed
cell volume was calculated
after the volume of medium
trapped
among cells was corrected.
The Ht value of the cell suspension
was determined
after correcting the trapped
medium
volume.
For the GA-treated
PH cell
was observed
in our
IMT,
cone-plate
Olympus
MS-230,
at 25 #{176}C
with
laboratory”#{176}
Co.,
(Model
Sugawara,
a cone-plate
combining
Optics
viscometer
(Model
dextran
to 55%),
solutions
and
an inverted
Tokyo),
a transpar-
B, Tokyo
Tokyo).
Keiki)
Two
and
a
experimental
(6%
a constant
or
10%)
shear
rate
or plasma
(752
with
s’)
was
varying
Ht
applied.
The osmolarity
of dextran
solutions
was adjusted
with NaCI
by
using a Halbmikro
osmometer
(Type
M, Knauer,
FRG)
after
dissolving
an appropriate
amount
of dextran
in a hypotonic
HEPES
buffer (50 mmoh/L
of HEPES,
5 mmol/L
of KCI, 6 mmol/L
of
glucose). The degree ofdeformation
ofstably
oriented and deformed
cells was expressed
by the DI, defined
by DI = (L - B)/(L
+ B),
where L and B are the lengths of long and short axes of ehlipsoidally
deformed
cells.3 For each sample,
DIs of 60 to 80 cells were
measured.
Measurement
of Viscosity
To measure
the viscosity
of hemolysate
and cell suspension,
a
cone-plate
viscometer
(Model
E, Tokyo Keiki) was used at 25 #{176}C.
For measurement
of intracellular
viscosity,
the hemolysates
of both
fractionated
Determination
Deformation
protocols were used as follows. First, cells were suspended
in isotonic
dextran solutions to make a Ht of0.4% to observe cell deformation
in
a diluted suspension.
The shear rates were changed
stepwise in the
range of 75.2 to 752 s’. The dextran concentration
was varied in the
range of 7.5% (4.0 cP) to 20% (18.3 cP). Second, for observation
of
the cellular deformation
at higher Hts, cells were suspended
in either
(4%
low-density
dicarboxyhic
with
Cells
Cells)
fractionated
ide
Hardened
Measurement
intact
cells
and
PH
cells
were
prepared.
cells were suspended
in 3 vol of hypotonic
mOsm/kg,
5 mmol/L
of HEPES,
10 mmol/L
After
membrane
components
were removed
(10,000
g for
ultrafiltration
nefilter
tion
GmbH,
of the
minutes),
agreed
the
Gottingen,
bag;
FRG)
reached
of original
with
hemolysate
a collodion
hemolysate
concentration
cells
20
(using
the
was
SMI3200,
until
that
packed
concentrated
by
Sartorius-Membra-
the hemoglobin
of the
concentra-
intracellular
cells. The viscosity
reported
The
HEPES
buffer
(30
of NaCI; pH 7.4).
by centrifugation
hemoglobin
of hemolysate
of intact
value.”
RESULTS
Characterization
To
and
ofPH
facilitate
to avoid
prepared
the
optical
the decrease
in
the
hypotonic
Cells
transparency
of internal
dextran
of cell
viscosity,
solution.
suspension
PH
cells
Intact
were
cells
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ABC DEFORMATION
resisted
IN SHEAR
hypotonic
Thus,
hemolysis
to enhance
By suspending
LPC-treated
concentrations
PH cells
could
membrane
the
Although
LPC
concentration
cholesterol
and
pretreated
in the hypotonic
After
reseahing,
be
progressive
increasing
cells
by washing
could
easily
loss
of dextran.
with LPC.
presence
were
the
removed
shape
the
cells
of dextran,
be varied.
was
cell
in
hemolysis,
different
729
FLOW
internal
with
of
of
solution
materials
been
2
0.4
C”
disc.
caused
reported,22
to the
flat
ellipsoid
cells, heavy
the properties
I , together
with
for comparison.
To
with
unequal
axial
ofCells
in Diluted
influence
and
rate
on the degree
of cells
trations
suspended
of dextran
various
shear
rates.
Fischer
DI of intact
cells
are
shear
and
rate
study,
lengths
medium
taking
the
cells.
a is the
The
1 A. At certain
DIs,
where
points
the
t
plots
light
and
respectively.
In
into
account,
where
,i,
is the intracehlu-
PH
cells,
heavy
Intact
with
were
.
I Thus,
in spite
various
shear
are
shown
in Fig
ated with
viscosity.
shown
density
(n5/n)
the
read
same
slope
plot
at the crossing
DI is expressed
in
1 . Hematological
Indices
line,
aging.
viscosity
Therefore,
and
cells
V
high-density
log
cells
(not
(‘i/ii)
as that
shown).
known
were
to
fraction-
of intracellular
for both low-
for PH cells
The
curve
in the
of DI v
of intact
cells slightly
shifts upward
(Fig 2B)
with that of PH cells (dotted
line is the curve for
shown in Fig 2A). To fit these curves on the same
another
fi
parameter
of PH Cells
must
and Fractionated
Intact
be
introduced;
DI
is then
)06#{149}Adopting
should
/3 = 1
to 0.7.
be reduced
Cells
DIt
,1,t
Cell)
is well
intact
expressed
as a function
of fl(t,/i
for the intact
cells, /3 for PH cells
t
and Deformability
(mmol/L
(am’)
Intracellular
cell
0.6
PH cells
[Hb].,’
.5!_
Cells
cells
as compared
PH cells
and all
of the difference
log (qexIin)
Percoll
to minimize
the variation
The same
value
of a was obtained
log ‘)“1
n(/m)
as a
PH cells.
Table
cells.
with
at
v log
-yi
lines
1
0.5
0
(sec)
is
increase
In Fig 2A, all DIs of light
are plotted
against
-‘y0x(05/tj)#{176}6’
curve
i
the
parameter.
of log
d of Fig
that
y and
1000
Fig 1 .
(A) Dependence
of deformation
index
(Dl) of light
partially
hemolyzed
(PH) cells on the shear rate in the isotonic
dextran
solutions.
Dextran
concentration:
7.5% (i,.
4.0
cP), 8.6%
(V. 4.7 cP), 10% (#{149}.
5.1 cP). 11% (A. 6.1 cP), 12% (.
6.7 cP), 14%
(V.
8.8 cP). 16% (0. 11.5
cP). 18% (& 14.5 cP). and 20% (0. 18.3
cP). Dl is represented
by mean
± SD of 60 to 80 cells.
Lines a
through
d represent
Dl = 0.40. 0.31 . 0.23. and 0.1 6, respectively;
‘
values at each DI value were estimated.
(B) Logarithmic
plot of
shear stress
(‘i”i.,)
v (suspending
medium
viscosity/intracellular
viscosity)
(n.,/m)’ Data and symbols are the same as in A. The
points on line a give a Dl value of 0.40, on line b a Dl value of 0.31.
on line c a Dl value of 0.23, and on line d a DI value of 0.16. The
lines are drawn
by linear regression
analysis
(mean
± SD of these
slope
-0.60
± 0.03).
DIs
shown
where
viscosity
y values
a through
the
1
I
I
500
coby
suspending
have
ylStO#{149}2
medium,
straight
lie on the same
between
of light
parallel
function
of /)O.6
and heavy PH cells
points
OTt 0
of various
concenwere
measured
at
to
medium,
adjustable
interpolated
on lines
of
of suspending
I B give
( -0.6),
DIs
viscosities
of
colleagues3
intracellular
and
in Fig
0.lj:
flow
Suda
and
are expressed
viscosity
viscosity,
lar viscosity,
and
were
cells
in shear
by
solution
0.4%)
and
.y’5o#{149}2
is modified
of the suspending
PH
light,
of these
ofdeformation,
is a function
the variable
the viscosity
rates
of
in the isotonic
(hematocrit,
the
present
and
Suspension
the
shear
.
‘a-
0.21-
1.
investigate
medium
>‘
C
no loss of
cells or
data about densityPH cells deformed
as well as intact
cells did, as reported
workers.2#{176}Thus, the degrees
ofdeformation
Deformation
C
V
d2
of PH
about
in Table
intact
cells
DI inTable
U
0.3
by
at a low
such as was used in the present
study,
phosphohipids
of either
LPC-treated
study,
two kinds
The typical
data
are summarized
fractionated
E
,.
PH cells was detected.
In this
prepared.
B
a
and
to biconcave
of membrane
has
solution
viscosity
in the cell
BSA
reverted
concentration
LPC
0.5
(cP)
j
-
150 s’
j
- 376
s’
PH
Light
96.0
2.58
3.5 (2.8)
0.35
±
0.05
0.47
±
0.02
Heavy
75.2
3.73
9.8 (5.0)
0.27
±
0.05
0.41
±
0.04
§
Intact
PH,
Low
density
89.0
5.05
10.3
0.31
±
0.04
0.44
±
0.05
High
density
81.5
6.20
20.4
0.22
±
0.05
0.36
±
0.05
partially
‘Intracellular
hemolyzed;
hemoglobin
tlntracellular
viscosity.
as that of PH cells.
Deformation
§lntact
cells
index
were
MCV.
mean
corpuscular
volume.
concentration.
The value in parentheses
(DI) was measured
fractionated
by density
is the viscosity
at shear rates
gradient
of hemolysate
(‘y) of 1 50 and 376
(described
in Materials
(prepared
‘
and
when
Methods
from intact
cells were
section).
cells) having the same hemoglobin
suspended
in 20%
dextran
solution
concentration
( 1 8.3 cP).
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
730
KON.
A
0.5
Oh
0.3
0.3
AND
SHIGA
A
0.5
014
MAEDA.
0
0.2
0.2
01
0.1
0
0
0
200
100
TQex
0
100
0
200
(A) Plot of deformation
index (DI) v
OS of
hemolyzed
(PH) cells. Light PH cells (0) were suspended
in
isotonic
dextran
solutions
of 7.5% (4.0 cP) to 20% (1 8.3 cP). and
heavy
PH cells (I) were suspended
in solutions
of 1 1 % (6.1 cP) to
20%. DI was measured
at shear rates of 75.2, 1 50, 376, 752 s’.
Data of light PH cells are the same as in Fig 1 A. Dl is represented
by a mean of 60 to 80 cells.
(B) Plot of Dl v ‘‘tps,,,#{176}’
of intact
cells. Fractionated
low-density
cells (0) were suspended
in isotonic dextran
solutions
of 14% (8.8 cP) to 20% (18.3
cP). and
fractionated
high-density
cells () were suspended
in solutions
of
16% (10.8 cP) to 20%. DI was measured
at the same shear rates as
in Fig 2A. The dotted
line is the regression
curve
for PH cells
shown in Fig 2A. DI is represented
by a mean of 60 to 80 cells.
Fig
Fig 2.
fact implies
that
cells; presumably,
impaired
during
of PH
cells.
induce
cross-linking
lar deformabihity.’5’23
is applied,
direction)
PH cells are less deformable
than
the PH cell membrane
would
be
preparation
Diamide-treated
fled.’5
Therefore,
membrane
proteins
DIs of diamide-treated
solution
from
15.5%
parameter
same
intact
)
curve
shown
curve
cells
cells
Light
PH
adopting
in Table
an
Plot
of
was
of
the
mmol/L
dependent
appropriate
2, all points
(no
on the
deformation
can
of 13 for
be brought
with the
13 decrease
on the
curve
as
ofdeformation
from
cells.
cells
in relatively
The
in 6%
rheoscopic
low
s’ are shown
in Fig 4 (line
becomes
clear as hematocrit
Ht range
flash
(4%
are plotted
suspension
(DI)
v
for
)‘
against
viscosity,
B). The ellipsoidal
increases.
This
of
at y
deformamonotonous
=
suspension
25%).
=
Therefore,
deformation
manner
of the dextran
as the
increase
On the other
of viscosity
hand,
viscos-
When
(Fig 5), where
ij
lie on the DI v ‘yncx(ne,/j,)#{176}6
DIs
the
DIs
is
the
curve
increase
of
in the same
solution.
at higher
h-Its (>25%),
DI measurement was impossible
due to cell crowding.
At a I-It of 45%,
however,
cells were photographically
observed
to deform
to
flat ellipsoid
but variously
distorted
shapes
and orient
with
their long axes parallel
to the flow direction
(Fig 4, line B).
PH
cells.
for
tion
ellipsoidal
PH
not
high
was
/)O.6
The
heavy
(eg,
to the
cells.
higher
compared
deformation
viscosity
with that of light
PH cells.
2.
ft Values
a and
the
to induce
dynes/cm2
intracellular
Table
could
Presumably,
enough
19
at
of
such
a Ht
heavy
of
cells
a
f
1
0.55
27.4
0.5
0.7
0.65
33.3
0.35
0.5
0.95
46.7
0
0.4
concentration
for diamide
treatment
polymerization
of intact
(described
due
as
Cells
0.6
tPercentae
of spectrin
Methods section).
of
deforma20%)
0
‘Diamide
not
value
PH
of Dia mide-Treated
Cross-linking
1%)
0
photographs
to increased
4%) to 4.2 cP(Ht
=
of light PH cells shown
in Fig 2A.
suspension
viscosity
acts on cellular
for
the
Dextran
to 25%)
is due
3.4 cP(Ht
Heavy
cells treated
/3 is fixed at
value
increase
Diamide’
(mmol/L)
PH
index
fi
be observed
proceeds.
Cells Suspended
Hts
200
(dyn/cm2)
diamide-treated
cells.
(A) The parameter
a is varied whereas
is
fixed at unity.
Fractionated
low-density
cells were treated
with
diamide.
Diamide
concentration:
0 mmol/L
(0). 0.55 mmol/L
(s).
0.65
mmol/L
(a). 0.95 mmol/L
(A). Cells
were
suspended
in
isotonic
dextran
solutions
of 1 5.5% (1 1 .0 cP) to 20% (18.3 cP). and
DI was measured
at shear rates of 75.2. 150. 376, and 752 s’; a
values
for cells treated
with 0. 0.55.
0.65.
and 0.95
mmol/L
of
diamide
are 0.6. 0.5. 0.35. and 0. respectively.
DI is represented
by
a mean of 60 to 80 cells.
(B) Both parameters
a and
are altered.
Data and symbols
are the same as in Fig 3A;
values
for cells
treated
with 0. 0.55. 0.65, and 0.95 mmol/L
of diamide
are 1 . 0.7,
0.5. and 0.4. respectively
(a values are the same as in Fig 3A). The
dotted
line is the regression
curve of DI v j’i#{149},(s#{149},/s,,)#{176} of intact
cells shown
in Fig 2B. The degrees
of cross-linking
of spectrin
of
cells treated
with different
concentrations
of diamide
are shown in
Table 2.
ity,
cells were measured
in the dextran
(11.0
cP) to 20% (18.3
cP). The
(Fig 3B), which
curve agrees
(Fig
2B),
ie, both
a and
Deformation
ofPH
Solution
at Various
752
tion
to <I
with appropriate
a values
for
of various
concentrations
whereas
Furthermore,
cross-linking
light
to
to the flow
is modi-
of cross-linking
on deformation,
be lowered
a for diamide-treated
diamide
unity.
each
shown
of cross-linking,
as estimated
from the plot of log -y
(not shown).
In Fig 3A, DIs are plotted
against
fln(n/m
with
been
parallel
hemoglobin
the influence
spectrin)
concentration
ofdiamide
must
buckling
pattern
was detected).
degree
V log
has
pattern
(furrows
and, in addition,
to study
(mainly
3.
100
rl.ex/rljn)
cells.
Diamide
of membrane
proteins
to decrease
celluWhen
a high concentration
of diamide
a buckling
appears24
2000
I3tri.ex(
(qex/rtin)#{176}(dyn/cm2)
partially
This
intact
100
cells.
in Materials
and
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
RBC
DEFORMATION
IN SHEAR
731
FLOW
Fig 4.
Deformation
of light
partially
hemolyzed
(PH) cells in the suspension
of
various
hematocrits.
Cells were suspended
in 10% dextran
solution
(5.1 cP, line A). 6%
dextran
solution
(3.3 cP. line B). and plasma
(1 .8 cP. line
C). Cell
deformation
was
observed
at a shear rate of 752 s’. Hematocrit
(Ht) of the suspension
ranges
from
4%to45%.
Deformation
Solution
ofPH
Cells
at Various
Hts
Light
PH
demonstrate
with
those
shown
.(fl/)O.6
in
Suspended
in 10%
in 6% dextran
4 (line
solution
A).
however,
despite
Ht
cells.
In 10% dextran
solution,
advanced
ellipsoidal
deformation
Fig
Dextran
When
DI
light PH cells
as compared
at lower
Hts
DIs
plotted
decreases
are
from
(<25%)
0.28
as
0.23
H
O.2
Heavy
PH cells.
as shown
of light
0Ti
1
0
1
50
(
., r#{149}lwr-t1/rt
)0.6
100
of PH cells
#{176}#{149}#{149}
shown
in Fig 2A.
60 dynes/cm2
(at
Ht
20%,
=
(at
,
=
Although
the intracellular
at
viscosity
was
was induced
due to high
In the Ht range of 4% to
in Fig 5, DIs lie on the DI v pje5(;,/tin)
PH cells shown
in Fig 2A. The suspension
changes
from
cP (Ht
5.9
to 7.6
4%)
=
cP
0.6
(Ht
Deformation
Hts
ofPH
Light
PH
the
lower
at
cells.
Cells
deform
at which
light
PH
cells
to variously
at Ht
cells
deform
in 6% and
45%,
=
10% dextran
Ht range
at Various
distorted
distorted
to the
PH cells.
Although
( I .8 cP), cells deformed
at the higher
in Plasma
however,
is conspicuous
C),
shapes
Suspended
(<25%);
Ht
(line
Heavy
was low
Cells
discs
ellipsoidal
as shown
same
in Fig
shape
as that
4
of
solutions.
the
extracellular
but distorted
(35%,
45%,
and
viscosity
in various
55%).
(dyn/cm2)
Fig 5.
Effect
of increasing
hematocrit
of partially
hemolyzed
(PH) cell suspension
on deformation
index (DI). Light PH cells were
suspended
in isotonic
dextran
solutions
of 6% (3.3 cP. 0) and 10%
(5.1 cP. ).
Heavy
PH cells were
suspended
in 10% dextran
solution
(A). Deformation
index (Dl) was measured
at a shear rate
of 752 s-i. Hematocrits
of the suspension
of light PH cells in 6%
dextran
solution
were 4%, 8%, 1 5%. 20%, and 25%. Those of light
PH cells in 1 0% dextran
solution
and heavy PH cells in 1 0% dextran
solution
were 4%. 8%, 1 5%, and 20%. DI is given as mean ± SD (n
60 to 80). The dotted
line is the regression
curve
of DI v
‘i”i.,(n../m.,)
to 80 dynes/cm2
20%).
deformation
0.1
from
/)O.6
high,
the ellipsoidal
deformation
viscosity
of the suspending
medium.
viscosity
0.31-
in
5.6 cP)
=
as shown in Fig 5. In Fig 4 (line A), a photograph
Ht = 45% is also shown,
in which
distorted
ellipsoidal
deformation
is observed.
20%,
curve
r
increase
71,
7.1 cP)
against
to
the
4%,
=
Deformation
Undeformable
oflntact
Cells
To investigate
Cells
the influence
behavior
ofdeformable
fractionated
low-density
with
either
cells)
a larger
or GA-treated
GA, undeformable
observed.
oriented
GA-treated
randomly
in the Coexistence
of undeformable
cells in shear
intact
cells
amount
PH
cells),
flow,
(tracer
of light
cells
(light
PH
cells
on the
amount
of
was mixed
PH
cells
(deformable
cells
treated
could
flow.
cells
a small
cells)
PH
and deformation
in shear
of
Cells
not
with
of tracer cells was
deform,
were
and
suspended
they
in
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
KON.
732
16.7%
dextran
cells
was
and
GA-treated
6.3%.
kept
On
(12.7
(0.4%),
PH
the
was
(Fig
the
varied
the
Ht
of tracer
Ht of light
intact
PH
from
tracer
The
ellipsoidal
suspension
(Ht
2.1%)
=
PH cells
the ellipsoidal
(Fig
was
6E).
2. 1% to
cells
the
of
cell
PH
amount
of
(to Ht
remarkably
=
disturbed
DISCUSSION
Increased
viscosity
the
deformation
of suspending
the
by intracellular
for various
present
on the
function
of
study,
)
applicable
(Fig
2A,B).
Taking
/3
to 0.7
for PH
cells
of PH
intact
intact
light
cells, indicating
that PH cells
cells. A remarkable
difference
and heavy
PH cells is detected
area
ratio
which
Fig 6.
(fractionated
cells
on the
of these
lipids
through
is one
cells
same
remains
is detected).
the change
of the
however,
Deformation
low-density
in
curve,
DI
that
of fractionated
PH cells are close to that
2A)
suggests
the curves
SHIGA
that
such
DI v
fi of PH cells is
stiffness
caused
in
of light
PH
cells
cells,
Dis
of light
how-density
of fractionated
low-density
is
cells at
stresses
(Table
1 ), because
the effect of lowered
viscosity
of PH cells on DIs is canceled
by the
given shear
intracellular
increased
membrane
stiffness.
Age-related
changes
in membrane
reported.”42627
In this
viscoelastic
study,
cells
the
have
no significant
age-dependent
changes
effect
in
on the
properties
no difference
curve
between
low-density
was detected
(Fig 2B). This
that
to
The
remarkable.
of cells,
=
of
a
=
in the
cells
result
and
may
membrane
ellipsoidal
treated
with
reduced
(Table
plot
constant
relation
DI v
highimply
properties
deformation
of intact
cell
cell) in mixed
suspension.
Fractionated
low-density
cells
(tracer
cells)
were
suspended
in
16.7%
dextran
solution
(12.7
cP; the
hematocrit
of the
tracer
cells
was
adjusted
to 0.4%)
with either
light partially hemolyzed
(PH) cells or glutaraldehyde (GA)-treated
PH cells. The deformstion of tracer
cells was observed
at a
shear
rate
of 752 s’.
(A) Only tracer
cells
present
in the
suspension.
(B
through
D) Tracer
cells are mixed
with
light PH cells. Hematocrits
oflight
PH cells
in the suspensions
of B. C. and D. are
2.1 %. 4.3%.
and 6.3%.
respectively.
(E
through
F) Tracer
cells are mixed
with
GA-treated
PH cells. Hematocrits
of GAtreated
PH cells in the suspensions
of E. F,
and G are 2.1 %, 4.3%.
and 6.3%. respectively.
DI
v
V
expressed
by the
brane,
ie, elastic
0.6
discocytes
no loss
No
be
)
The
in
ie, the partial
in a. The parameter
modulus.
According
dimension
of
in Fig
of
the
shear
result
hemolysis
causes
(dynes/cm’Y’.
of memand mem-
compressibility
method,
but
because
strongly
resist
demonstrated
modulus
but
may correspond
i3 may thus
to dimensional
be
in the
is physically
area
Meiselman
must
3).
present
area
are
curve
material
constants
compressibility,
at constant
and
fi,
a and
shown
is
density)
of the membrane
in
easily
proteins
low
to the same
alteration
in area.’#{176}Nash
result,
(as
detectable
the ghost
has a greater
brane
viscosity.”
Their
(of
parameters,
three
intrinsic
modulus,
area
deform
change
cells
all data
property
not
1 for the
to fit the
two
fr)’715(?1/Th
mechanical
of membrane
intact
2) to bring
viscosity.28-29
the cellular
(S/V)
of deformability.
of DI
the
diamide,
brane
of
(because
of cross-linking
When
would
v
This may affect
of surface/volume
influence
is a
stiffDI
are less deformable
than
of cell volume
between
(Table
1 ), whereas
the
determinants
the
effect
that
kinds
be reduced
(Fig
not shift
in membrane
integrity.
the intracellular
viscosity
half
The
membrane
revealed
For various
data
difference,
about
PH cells
does
Therefore,
the decrease
to the increased
membrane
due
by changes
Although
of
Pfaffe-
the
and
and
cells,
of membrane
deformation
viscosity.
demonstrated
intact
surface
f should
degree
stresses.
we
heavy
ratio
m)#{176}6
throughout.
of intracellular/extracellu-
deformation
fl’Yex(’iex/in
known
the
the differences
between
eliminated
if the DI was
viscosities
cellular
is unanimously
ratio
is well
although
that
were
shear
extracehlular/intracellular
ness
medium
is influenced
of the
Membrane
and
AND
shear flow.
of RBCs,
as a function
lar viscosities
and
deformation
rott and co-workers25
showed
young and old cell deformation
In
of S/V
density
Contribution
ofintracellular
Viscosity
Stiffness
to Cellular
Deformation
plotted
light
change
were
6F,G).
enhance
between
presumably
PH cells
to higher
however
was
could
light PH
GA-treated
When
increased,
deformation
cell
deformation
not affected
in the deformable
6B,C,D),
nor in the diluted
the GA-treated
(Fig
the
suspensions
concentration).
intact
cells
suspension
in which
and
from the light P1-I or GA-treated
of cell shape
and of darkness
(due
hemoglobin
6.3%),
cell
cP)
flash-photographs,
be distinguished
by differences
cell
solution
constant
MAEDA.
that
a similar
memto the present
the change
in /3 but not
be related
to the shear
analysis,
fi should
have a
Therefore,
a reciprocal
of /3
would
express
the relative
shear
modulus,
and the shear
modulus
of PH cells may be = I .4 times higher
than that of
intact
the
cells.
shear
The
diamide
modulus24
treatment
and
may
is also
cause
known
increased
to increase
membrane
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
RBC
DEFORMATION
viscosity’2
may
IN SHEAR
by
restricting
the
head to the decrease
733
FLOW
flexibility
of two
of cytoskeleton.
parameters,
present
This
a and
/3.
result
tion
of
cells
increasing
Ht Dependency
The
of Cellular
shear
corresponds
the
rate
applied
the
calculated
to
capillary
(-.1,000
behavior
cells.
s’),
of PH
Therefore,
possible
up
to
PH
the
Ht
solutions,
respectively,
increase
of suspension
mation
-20
m)#{176}#{149}6’
the shear
tion.
value
On
the
that
at
this
decline
is not
have
enough
the
value
critical
h-It
that
the
value,
a
in
same
as that
the
Ht
At
as
The
Ht is shown
cell
crowding
the
effect
higher
5. In
disturbs
of Ht,
Kon
is increased
viscosity;
the
effect
the
the
cells
was
progressive
ghost
at
the
to ellipsoid
PH
be due
to the
of cells
shear
discs
stress
Kon
study
a and
and
to
internal
viscosity
viscosity
(i/);
cell
There-
(a) The degree
shear
Ht
generally
of
stress
(reflected
of suspending
and (b)
by raising
(i)
and
intact
cells is maintained
cells.
that:
ratio
intact
coworkers.’6
properties
the
conse-
on
on
by the applied
membrane
3), and
of laminar
which
flow is known,4
cells
reveals
PH
suspension.
acting
of hardened
by
in light
cell
cells,
in Poiseuille
is determined
the viscoelastic
of
suspension
disturbance
hardened
effective
the present
by
but to irregularly
concentration;
thus,
deformed
reported
described
deformation
cell
in GA-treated
effect
was
on the
medium
increasing
enhances
cellular
deformation,
but above a certain
Ht deformation
suppressed.
At physiological
Ht, cellular
deformation
is
is
mainly
of
influenced
extraceliuiar
medium
(>25%),
with
collision
between
cells induces
In the present
study,
the frac-
of the
disturbing
deformation
and
the
on
fore, the ellipsoidal
deformation
of intact
by ellipsoidal
deformation
of surrounding
suspension
begins,
extracellular
Hts
a similar
viscosity
ie,
operative
in the
the
not
of rigid
parameters
a
cells
may
Tumbling
and
above
dextran
concentration
by tumbling
reduces
(-“i)
the
solutions
ie, the
cells
but
phenomenon
In short,
rather
deforma-
10%
not deform
to ellipsoid
with increasing
ghost
low-density
deformation
of deformation
Ht
by Ht,
medium
and
the contribution
of viscosity
is not significant.
quantitative
even with PH cells. The
in plasma
is qualitatively
10% dextran
ellipsoidal
by Fischer
cells.
in
cell
have proposed
that
irregular
deformation.
suspension
than
is
cells did
ellipsoid)
quently
deforma-
cellular
induced
and
Marlow’5;
of intact
flow caused
in
in Fig
suppressing
higher
in 6% and
45%, however.
This
suspended
as shown
measurement
of DI is not possible,
degree
of deformation
of PH cells
the
in
is smaller
extraceilular
which
lower
viscosity.
increase
deformation
the
on
at
cell
The
progressively
becomes
of
and
tionated
to
is
in 6%
mechanism
amount
they
such
due
the
PH cells
degree
as
depending
toward
increases
to induce
Concerning
is suppressed
certain
The
and
dextran
is below
ellipsoidal
because
shown
light
10%
of
60 dynes/cm2
lines.
(intact
distorted
to increasing
Ht or dextran
degree
of ellipsoidal
defor-
Dl of light
the
probably
on RBC
is
as follows:
plasma
but
influence
of intact
and heavy PH cells suspended
because
at low value of
hand,
phase,
orientation
the
80 dynes/cm2
stream
shifts
either
same
and
deformation
and
is observed.
at
suppressed,
6%
The
Goldsmith
tracer
deformation
the value
force
cell
When
in
viscosity
(/)#{176}6
uniform
-20%.
progressive
other
I 0% dextran
Kon’4
of
both light
cannot
deform,
dynes/cm2,
to that
in
Ht.
and
flow”
information
of PH
suspended
5). However,
medium
gives
higher
s’)
in artery
Poiseuille
is similar
study
752
=
rates
that
in flow
viscosity
owing
induces
the
(Fig
any
cells
(‘y
shear
assuming
are
study
wail
Ht,
suppressive
this
flow.
measurement
cells
suspension
concentration
in
the present
behavior
in blood
The quantitative
heavy
Deformation
is explained
PH
solutions
ACKNOWLEDGMENT
at a Ht of
deformation
of intact
cells at a
and Schmid-Sch#{246}nbein.2
The
We wish to thank
and Misuzu
Sekiya
assistance
with
Professor
and
Syoten
Takuya
experimental
Oka
for valuable
Taniguchi
for
their
discussions
technical
procedures.
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1987 69: 727-734
Erythrocyte deformation in shear flow: influences of internal viscosity,
membrane stiffness, and hematocrit
K Kon, N Maeda and T Shiga
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