From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
B L 00
DECEMBER,
D
T
of Hematology
VOL.
1968
Physical
Properties
in
Vivo.
as
I.
of
By
Red
by
Viscosity
HALE
Increased
increased
OF STUDIES
that
indicate
rigidity
F.
R. Murtr’rry
on
used
to describe
a shift
to more
to
rigidity
of cells
ing, hypertonic
report’
thermal
to
increased
Ricuiw
DUNN,
changes
in their
W.
developed
in
the
increased
destruction
that
increased
rigidity
microcirculation
spleen
changes
Newtonian
in red
type
the
Supported
and
W.
R.
heated
cells
will
result
of red
report2
of Medicine,
(C9) from
cells leading
of flow
and
to
lack
force
or by diminproperties
will be
to possible
effects
is compared
is
concerned
cells.
the
with
the
A hypothesis
in abnormal
beds,
to
or formaldehyde,
sicklshrinkage.
In a second
of hemolysis
is related
such
as
is
blood
those
flow
of
the
HAM,
19, 1968;
M.D.:
of the
Reserve
School
accepted
for publication
Case Western
the National
July
Reserve
Institutes
University.
of Health,
23, 1968.
Hanna
Payne
Professor
of Medicine,
Department
of
Division
of Research
in Medical
Education,
School
of
University.
REBECCA
F. DUNN,
B.A.: Research
Assistant
of Medicine,
Case Western
Reserve
University.
RIcHARD
Formerly
student,
School
of Medicine,
Case Western
Reserve
University;
of Radiology,
Stanford
Medical
Center,
Palo Alto, California.
JoHN
M.D.:
Associate
Professor
of Medicine,
Department
of Medicine,
School
of
Department
MURPHY,
BLOOD,
of
of
M.D.:
SAYmI,
Medicine,
vivo
Department
of Medicine,
School
by Research
Grant
HE-01263
and Director
Case Western
of Medicine,
presently,
third
circulatory
HALE
Medicine,
Medicine,
Department
in
A
properties
deformability.
kidney.
U. S. Public Health
Service.
First submitted
February
THOMAS
cells
in
special
and
From
rigidity.
of
SAYRE
in physical
rigidity
or
fixation
with
glutaraldehyde
and combined
sickling
and
of red
cells
or products
and
viscosity
mechanism
by
Hypertonicity
and increased
blood
destruction.
the rigidity
of unmanipulated
following
shrinkage
treatment
6
Effects
Altered
of deformability
as measured
by packing
under
centrifugal
ished
flow through
microfilters.
Alterations
in these
physical
related
to changes
in morphology,
hemoglobin,
stroma
and
on the microcirculation
In this first report,
NO.
Erythrocytes
Cells
and
Jom.
is reported
abnormalities
is a term
of flow,
viscosity
of
REBECCA
AND
SERIES
red cells
of
Sickling
HAM,
XXXII,
as Related
Rigidity
Fixation,
THOMAS
Cells
Increased
Measured
Chemical
A
he Journal
Case
VOL.
32,
Western
Reserve
No.
6 (DECEMBER),
University.
1968
847
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
848
HAM
MATERIALS
AND
ET AL.
METHODS
Blood
Nonnal
human
5 per cent CO2
coat were removed.
were
washed
twice
of
7.4.(3)
dog
in
air,
In
at
blood
to
some
C.
4
was
maintain
defibrinated
the
three
in
volumes
flasks
between
the
experiments,
in
pH
serum
of
containing
7.35
and
was
replaced.
7.5.
a
in
Chemical
of
red
serum
In
buffered
phosphate
gaseous
The
cells
of saline
mixture
and
bulfy
the
others,
isotonic
at pH
8, blood was defibrinated
in an open
solution.
In studies
of red cells in hypertonic
was
mixed
with solutions
of sodium
chloride,
urea,
or a combination
of
final
concenfration
of 10 per cent
by volume
of the aqueous
phase
and
hypertonicity
when combined
with the serum-red
cell mixture.
twice
In studies
3 volumes
or
cells
at pH
washed
NaC1
flask and
media,
the serum
these,
to give a
a predetermined
Fixation
Glutaraldehyde,
1 per
cent,
in buffered
0.6 per cent sodium
chloride
was
adjusted
to
pH 7.4 by titrating
with 0.15 M NaOH,
and kept at 4 C. protected
from light.
A mixture
was also made of one part of aqueous
formaldehyde,
40 per cent, and 9 parts 0.85 per cent
sodium
chloride
buffered
to pH 7.4. (4) The final mixture
had a pH of 7.2 and was kept at
room
temperature.
One part
of washed,
packed
red cells was suspended
in four parts
of
each fixative
and incubated
at 4 C. for 30 mm. There
was no difference
in fixation
when
the
ratio
of fixative
to packed
red cells was varied
over a range
of 1:1 to 1:19.
Chemical
fixation
by these
agents
is probably
achieved
by cross-linking
intracellular
and membrane
proteins.
Cell
volume
following
fixation
was
determined
by measuring
the dilution
of
1311 albumin
in suspensions
of fixed and control
cells.
Preparation
of Samples
Stroma-free
hemoglobin
solutions
were
prepared
in two ways
to give a range
of concentrations
from
2 to 40 Gm.
per cent.
In the water-hemolysis
method,
the washed
red
cells were
suspended
in 9 volumes
of water
and the pH was lowered
to 5.5 by bubbling
CO2 through
the mixture,
followed
by centrifugation
at 1,600
X C for
20 mm. at 4 C.
In the Singer
method,4
in most instances,
washed
red cells in 1.5 volumes
of water
and 0.4
volumes
of toluene
were mixed
vigorously,
centrifuged
10 mm.
at 3,000
X G at 25 C. and
the
toluene-stroma
layer
removed.
The
hemoglobin
solutions
were
recentrifuged
at
17,000
x G to remove
fragments
of stroma
and concentrated
by perevaporation,
blowing
an air current
across
cellophane
tubing
containing
the solution.
Red cell stroma
was prepared
in stainless
steel test tubes
(115 mm.
X 20 mm.)
by freezing washed
red cells
for 5 minutes
at -75
C. and thawing
for 6 minutes
at 37 C.
three
times.
The
preparations
were
washed
by suspension
in 9 volumes
of buffered
isotonic
NaCl
and centrifuged
at 17,000
X C at 4 C. Sonic
treatment
of red
cells or stroma
was
performed
in a Raytheon
10 kilocycle
oscillator
for 5 to 20 minutes
at 10 C. After treatment,
all samples
were
examined
by phase
microcopy
for the presence
of intact
formed
elements.
Reduction
done
in
of
a 250
blood
ml.
or
tonometer
stroma-free
by
hemoglobin
equilibration
for
solutions
30 minutes
in
10-15
in a 37
ml.
volumes
C. water
was
bath,
using
gaseous
mixtures
of 90 per
cent N2 and 10 per cent
C02,
to provide
a pH of 6.6 to 6.7,
or 90 per cent N., and 10 per cent CO.,, to provide
a pH of 7.2 to 7.3. Samples
were oxygenated
with gaseous
mixtures
containing
95 per cent
02
and
5 per cent CO2 in a comparable
manner.
Hemoglobin
concentration
was determined
by the cyanmethemoglobin
method.4
Hematocrits
were
determined
by the method
of Wintrobe
and
by the microhematrocrit
technique
with
centrifugation
for
8 minutes.4
Estimates
of
erythrocyte
deformability
or
rigidity
were
obtained
by determining
the flow of cell suspensions
in serum
or buffer
salt
solutions
Corporation,
through
Bedford,
microfilters5
of
Massachusetts.
approximately
8 and
9
s manufactured
by
the
Millipore
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
PHYSICAL
PROPERTIES
OF RED
CELLS
849
I.
30
SA
28-h
-
REDUCED
FIXED
.
CELLS
CONTROL
26-
22-
Fig.
1.-Viscosity
at high
rates
(Ostwald
viscosimeter)
I6
05
8
cells
mal,
form
I4
(1)
2-
:
--
,
.
HE MATOCR
Viscosity
Viscosity
measurements
Viscosity
a
differing
fixed
(sickle
hematocrits
and
cells
trait).
shear
red
for
in
the
nor-
sickled
-
.
cent)
Determinations
The rates of flow were
samples
of the various
medium.
Rates of flow
medium.
This
procedure
1,000 sec.-167
on
Cper
IT
at
of
determinations
Brookfield
tories,
at high
shear
rates
were
made
with
the Ostwald
viscometer.
observed
in a constant
temperature
water
bath at 37 C. using 5 ml.
suspensions,
comparing
the flow rate
to that
of the suspension
were expressed
as a ratio of that of the test substance
to that of the
gave
maximum
but
indeterminate
rates
of
shear
in
of
Inc.,
at
cone-plate
Stoughton,
Resistance
low
shear
rates,
viscosimeter,
at
model
1.15 to 230 sec.-1,
LVT
1/2 (Brookfield
were
37
C.
Labora-
to Packing
Mechanical
The
at
Massachusetts).#{176}
Whole
blood
was centrifuged
at room
temperature
in Wintrobe
35 minutes
at 1,500
x G, and for an additional
period
of 20 minutes
observations
at 5 or 10 minute
intervals.
The
data
obtained
were
line of the packed
red
cell volume
observed
by the
microhematocrit,
fugation.
et al.4
made
Engineering
hematocrit
at 3,000
X
compared
to
8 minutes
tubes
for
G, with
a
base
of centri-
Fragility
mechanical
The
fragility
hematocrits
of the
of
red
blood
samples
cells
were
was
determined
observed
but
not
by
the
method
of
Emerson
adjusted.
RESULTS
A. Effect
Red
normal
behavior,
of the
cells
Fixatives
fixed
cells
i.e.,
(Figs.
less
in glutaraldehyde
1 and
dependent
2)
or formaldehyde
and
on
the
shear
flow
properties
rate.
Suspensions
were
were
more
more
viscous
Newtonian
of normal
red
than
in
cells
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
850
ET
HAM
AL.
Sc
40
30
Fig.
2.-Viscosity
measured
at
low
shear
rates
(Brookfield
viscosimeter)
of
red cells fixed m glutaraldehyde
compared
to samples
suspended
in isotonic
sodium
chloride.
Two
levels
of hematocrit
are shown.
t,,._.
.
HEMATOCRIT
S
(SI FIXED)
20
#{149}
“
\
>
2
(41
25
50
00
75
SHEAR
characteristically
non-Newtonian
showed
behavior.
microfilters
whereas
such
filters.
Fixed
cells
untreated
minutes
were
and
not
cells
at similar
hemolyzed
x
3,000
were
appearance
examined
C, fixed
required
by
three
50
75
shear
rates,
did not flow
flowed
processes
200
that
that
is,
through
rapidly
through
readily
destroyed
vibration
for
cells maintained
10
weeks
in sealed
slide preparations,
microscopy.
When
centrifuged
to pack
the fixed
(Fig.
cells
3).
230
SEC1
higher
cells
thawing,
sonic
The fixed red
for many
by phase
red cells failed
to resuspend
at
concentrations
cells,
namely,
freezing
and
and repeated
washing
in water.
mal morphologic
in the room
and
tation
fresh
decreasing
viscosity
The glutaraldehyde-fixed
25
RATE
Nod)
Extreme
after
or 20
a norkept
1,500
at
degrees
of agiThere
centrifugation.
30
28
o...X
FIXED
.-.
CELLS
CELLS
IN
NoCI
26
7
24
22
Fig.
3.-Resistance
to packing
of red cells fixed in glutaraldehyde
compared
to those
in saline
when
centrifuged
in
Wintrobe
tubes
20
(8
I
at
6
different
intervals
of time and two
gravitational
forces.
The microhematocrit
method
at 13,000
x C for
8
the
mm.
control
was
used
level
0
3000
0
(2
for
measuring
of hematocrit.
z
6
#{149}
4
0 #{149}-__#{149}
5
(0
(5
TIME
25
OF
CENTRIFUGATION
35
45
(mInutes)
55
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
PHYSICAL
PROPERTIES
Table
1.-Effect
Normal
OF
on
Human
RED
CELLS
Hematocrit,
Blood
851
I.
Mechanical
in Combinations
Chloride
and
of
Urea
Fragility
of
Different
Concentrations
(Ostwald
of
NaCl
Urea
mOsm./Kg.
(dilution
per cent
H20
(change
10 per cent)
Done
per cent)
300
Viscosity
of
of
Sodium
Hematocrit*
After
in
and
fragility,
per cent
Hematocrit
Cells
Viscosinieter).
Mechanical
Concentrations
Red
60
minutes
immediately
per
at
\‘iscosity*
cent
medium=
(change
37C.
in
in
per cent)
per cent
40.5
4
3
39.5
2.9
300
300
40.3
2.5
3.5
38.3
2.9
300
600
40.2
4
3.5
39.2
2.9
300
900
40.5
4
300
1200
42.8
26.9(-32)
4.0(--38
600
1
(change
3
26.6(-34)
10
600
300
28.8(-29)
15
600
600
27.3(-33)
16
900
26.2(-34)
59
1200
23.0(-43)
94
14
18
26.6(-32)
4.0(+38)
26.6(
4.1(+39,I
-321
23.2(-41)
6.4(±
120)
(Hemolysis)
*These
were
observations
were
no signs
it was
done
at a different
of agglutination.
found
that
no
net
cells and the supernatant
volume
was unchanged
per
time
Using
from
the same
albumin
movement
solution
although
on blood
as a tracer
131J
of water
subject.
had
added
occurred
when
glutaraldehyde
the apparent
increase
to plasma,
between
the
was added.
in hematocrit
red
Thus
cell
was 22
cent.
B. Sickled
It
Red
has
solutions
already
been
from
homozygous
mation
when
homozygous
show
CelLs
viscosity,
mechanical
sickled
cells
formaldehyde
(Fig.
Suspending
as shown
changed
300
utes
cells
corpuscular
addition
300
than
and
on the
to 900
300
1 and
normal
red
mOsm./Kg.
water,
to 1,200 mOsm./Kg.
did not increase
mechanical
mOsm.,
the
in hypertonic
in Tables
when
In
hemoglobin
of urea,
fragility
1,200
that
form
a viscous
stroma-free
gel
the intact
red
disease,
when
to
packing
by
the
study
reported
viscosity
of cells
hemoglobin
with
centrifugal
force,
here,
fixed
tactoid
for-
cells of patients
in the sickled
the
with
form,
and
in-
viscosity
of
glutaraldehyde
with
or
1).
of Hypertonicity
C. Effect
mean
resistance
greater
Harris8
disease
Further,
sickle
cell
fragility.9”0
was
by
sickle
oxygen
is removed.
and heterozygous
increased
creased
demonstrated
Intact
Red
media
decreased
Cell
concentration
mOsm./Kg.
2. The
cells
of water,
mechanical
were
did
fragility
suspended
alone
or in the presence
of water.
Preliminary
the mechanical
fragility
of normal
cells
mOsm.,
increased
the
hematocrit
(MCHC)
not
and
in
serum
of urea
incubation
of those
in hypertonic
progressively
and
(Tables
affect
increased
1, 2, 3).
the
hematocrit
mixtures
The
hematocrit,
were
of
not
NaCl,
at concentrations
of
at 37 C. for 60 minmixtures
tested.
The
NaC1,
600 mOsm.,
and was
not affected
900
by
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
852
HAM
Table
2.-Effect
on
with
Hematocrit
Sickle-Cell
and
Trait
Mechanical
Fragility
as Observed
*
Sodium
in
Chloride
of Red
Different
and
Cells
from
Concentrations
of
NaC1
per
H20
(dilution
10 per
300
300
300
300
600
600
600
300
600
900
1200
*Similar
the
results
were
presence
of red
of urea.
cells
with
Phase
suspended
of urea.
Similar
with
sickle-cell
In
obtained
trait
oxygenated
and
blood
60
minutes
immediately
cent)
at
37C.
4.3
5.1
1.5
43
4.9
4.7
45.2
29.4(-318)
31
(-28.1)
21
29.5(-31.5)
27
26.5(-38.5)
81
26.4(-38.7)
74
from
microscopy
mOsm.
samples
Done
43.1
15
24
a patient
with
showed
no
21
homozygous
mOsm.
sickle
change
cell
in the
water
using
oxygenated
sickle cell disease
at 600
5.0
4.8
of NaCl/Kg.
were
observed
homozygous
results
After
in
42.6
blood
in 300
cent
cent
per
300
600
900
Fragility
per
(change
cent)
a Patient
Urea.
Hematocrit
Urea
mOsm./Kg.
AL.
of
Mechanical
Concentrations
ET
or upon
disease.
morphology
the
addition
blood
from
(Table
2).
patients
of NaCl,
some
red
cells
were
in
the form
of normal
biconcave
discs,
but most
were
crenated
with
about
an
equal
distribution
of irregular
(lumpy)
discs,
crenated
(berry)
cells and serrated
(burr)
cells.
These
morphologic
changes
occurred
indistinguishably
in
samples
of blood
from
normal
human
or canine
subjects,
or patients
with
sickle-cell
trait
forms
isotonic
regained
media
sickle
cell
(Figs.
4 and
their
at 300
disease,
5).
normal
mOsm.
there
No
sickling
biconcave
of NaCI.
always
was
observed,
were
permanent
sickled
sickled
cells
appeared
in hypertonic
solutions.
At
NaCl/Kg.
water,
spindle
forms
became
prominent
normal
human
and dog blood.
These
cells
seemed
points
often
resembling
sicided
cells.
There
was
Table
no change
3.-Effect
of
on
Sodium
in morphology
Viscosity
of
Chloride,
of normal
Deft
brinated
Comparing
Produced
Hematocrit
Concentration
of NaCl
mOsm./Kg.
(dilution
10 per
HmO
cent)
300
600
*Hemoglobin
Sonic
47.5
1,200
concentration
Cells
cells
cells
of
Increasing
with
Hemolysis
crenated
but
no
in isotonic
Concentrations
Products
Viscosity
(medium
=1),
per cent change#{176}
Sonic
Whole
Blood
disruption
33
3.1
31.5
(-34)
44
5.5
(+80)
2.1
25.8
(-46)
47
4.6
(+50)
2.1
15.6
Cm.
per
100
ml.
for
each
in
new
or hyper-
Disruption.
Mean
corpuscular
hemoglobin
concentration
of red cells
(Gm./100
ml.)
per cent
(change
in
per cent)
the
900 and
1,200
mOsm.
of
in all samples
including
flat and
shrunken,
with
red
Blood
Intact
by
and
disc-like
shapes
when
resuspended
In oxygenated
blood
from
homozygous
sample.
2.1
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
PHYSICAL
OF
PROPEIITIES
RED
NaCI
NORMAL
300mOsm.
BICONCAVE
600
NaCI
IRREGULAR
dS
DISC
mOsm.
(BERRY)
CELL
SERRATED
(BURR)
CELL
NaCI
SPINDLE
Fig. 4.-Diagramatic
sketches
of
characteristic
shapes
of normal
human red cells, dog cells, and cells
from
sickle-cell
trait
when
suspended
in isotonic
and
hypertonic
sodium
chloride.
No sickled
forms
were observed
in these oxygenated
samples.
DISC
(LUMPY)
CRENATED
4j
853
I.
CELLS
I200mOsm.
FORMS
p
tonic
media
when
oxygen
removed,
was
but
in isotonic
sickle-cell
trait and
form.
In 600 mOsm.
homozygous
of NaC1/Kg.
sickle
cell disease
water,
however,
crenated
sickle
produced
forms
of
length
and
few
sickled
tactoid
form.
The
formation
(crenated)
Viscosity
cells,
cells
in number,
appearance
occurred
human
cells,
gous
sickle
cell anemia
meter)
when
suspended
was
viscosity
NaC1
in
the
sickle
cells
added
for
showed
cells
6).
the
NaCl
rose
(Figs.
constant
viscosity
celLs.
was
viscosity
cent by
increased
6 and
as the
shear
7).
were
normal
than
tonicity
to 900
cells.
and
MCHC,
as the red cell
was also an increased
tubes
(Fig.
Viscosity
human
to 600
cells
was
mOsm.
volume
resistance
urea
had
of NaCl
progressively
to packing
no effect
further
shrank
(Tables
by centrifugal
red
homozyviscosiof isotonic
mOsm.,
the
in hypertonic
increased
(Fig.
viscosity
as the
indicating
that
had
a greater
on viscosity.
increased
in
classic
dog
and
(Ostwald
volume
Red
rate
short
Increases
viscosity
and
in
the
1, 2, and 3). There
force
in Wintrobe
8).
of
blood
Adding
1,200
of
evidence
that
the shrunken
trait
Conversely,
red cells in isotonic
media
showed
decreasing
shear
rate was increased.
The viscosity
curves
actually
crossed,
the cells in hypertonic
NaCl,
even
at a decreased
hematocrit,
the
cells
the
Oxygenated
7).
viscosity
red
into
sickle-cell
normal
10 per
cells
that
deformed
was indirect
hemoglobin
of
with
When
oxygenated
a nearly
patients
the
to the classic
sickled
of oxygen
from
the
processes
not
of oxygenated
from
showed
the same
in serum
to which
(Fig.
the
were
of sickled
processes
the concentrated
sickled
red cells.
and resistance
to packing
normal
NaCl
but
media
changed
removal
reduced
or dog
blood
blood
had
in
hypertonic
no effect
media.
on viscosity.
Deoxygenation
When
blood
from
of normal
patients
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
854
HAM
Fig.
5.-Phase
of 800
and
serrated
with
microscopy
mOsm.
of sodium
cells.
sickle-cell
concentrations
first
cosity
gel
per
forms
maximal,
because
gel
and
formation
decreased
tonicity.
normally
reduced.
Accordingly,
in hypertonic
The increased
reversed
when
returned
in 600
the
(serum
with
NaCl)
trait
and homozygous
mOsm.
NaC1,
described,
and
then
or became
behaved
occurred.
the
cells
showing
sickle
cell disease
was
elevated
to a maximum
was
cells
trait
of water
at
AI
a concentration
irregular,
crenated
occurred.
viscosity
as just
sickle-cell
kilogram
or homozygous
NaCl,
suspended
viscosimeter
the
oxygenated
of the 600 mOsm.
media
red cells from
sickle-cell
was
in which
sickled
trait
of
the effect
When
the
were
No
of
chloride
E
like
a gel.
Reoxygenation
viscosity
to
the
reduced,
This
at isotonic
exceeding
upon
normal
sickle
cell
was
cells.
disease
however,
unmeasurable
of
value
reduced
degree,
the
in the
the
these
samples
predicted
from
vis-
Ostwald
only
situation
eliminated
the
level
of
cells from
patients
with
sickle
cell hemoglobin
behaved
NaCl
when
oxygenated,
but
frequently
gelled
when
viscosity
of cells
in hypertonic
NaCl
was completely
to isotonicity.
Crenation
without
hypertonicity.
bated
at 37 C. for several
hours
When
in unbuffered
normal
human
red cells were
isotonic
saline
at pH 8, 75
incuto 100
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
PHYSICAL
PROPERTIES
6.rNOCI,
mOsm
OF
RED
CELLS
855
i.
/KgH2O
NORMAL
600
SA
300
600
mOsm
#{149}
NORMAL
‘5
Fig. 6.-Effect
wald
viscosimeter)
:
.‘
#{149}
#{163}
/
‘/
mozygous
sickle
viscosity
is plotted
served
hypertonic-
cell
from
nordogs
and
trait or hodisease.
in the
The
against
which
hypertonic
hematocrit
creased
6
(Ost.
viscosity
of
ity of oxygenated
blood
mal
human
subjects,
patients
with sickle-cell
#{163}
#{163}
on
the
ob-
was
media.
de-
300m0sm
;Ji
20
24
28
32
36
40
44
HEMATOCRIT(
per cent
changed
to
duced
by 600 mOsm.
hematocrit,
varying
from
40
Gm.
readily
remained
preparation
associated
per
and
in
During
to the
showed
a gradual
to those
change
significant
increased
Red
proin
crenation.
CelLs
from
normal
similarly
at
the
range
no
this
hemoglobin
behaved
cent.
appearance
was
Disrupted
trait
increased
fluid
with
and
of oxygenated
sickle-cell
10 and
was
comparable
4). There
Stroma
solutions
heterozygous
concentration
60
56
forms
(Fig.
or viscosity
of Hemoglobin,
Stroma-free
those
with
52
crenated
of NaCl
MCHC
Viscosity
D.
48
per cent)
process
of
of 32 to 34 Gm.
rise
in
subjects
concentrations
air
and
drying,
the
cent.
The
(Fig.
9).
per
viscosity
30
20
Fig.
a
5
in serum.
(52)
3
mOsm/eg.
NOO.
600
25
50
75
at
for red
and hypertonic
The
low
vis-
(Brookfield
cells
NaCl
in
number
of red
sample
is
H 0
cells
same.
2
300
2
rates
cosimeter)
isotonic
36)
6
7.-Viscosity
shear
HEMATOCRIT
00
SHEAR
25
RATE
‘50
SEC1
?5
200
230
in
each
the
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
HAM
856
24
NoCl.
ET AL.
m0sm/Kg
H.)0
900
A-A
22
600
20
0
8
0
,‘..
6
Fig
8.-Resistance
to packing
of blood
samples
at three
ton.icities
of NaCl
compared
at intervals
of
centrifugation
at two gravitational
forces.
3000
‘5000
‘A
0
300
0-0
0
‘4
N
2
0
I0
8
6
4
2
5
0
5
25
TIME
Above
36 Gm.
phane
tubing
cosity
carried
measurements
out promptly
concentrations.
per
but
cent,
these
precipitation
particles
detected
was
could
be
45
CENTRIFUGATION
on the
redissolved
could
be made
in the
and showed
an abrupt
At a concentration
OF
for
55
(minutes)
surface
of the
a brief
period.
celloVis-
Ostwald
viscosimeter
if these
were
rise (Fig.
9) as compared
to lower
of 40 Gm.
per
cent
or higher,
apparent
gel
30r
#{149}
SA
28]
#{149}
NORMAL
2#{128}.-
24-
22
20--
Fig.
9.-Viscosity
of stroma-free
(Ostwald
viscosimeter)
hemoglobin
solutions
concentrated
by air drying.
Gel formation was detected
above
36 Gm. per cent
and was marked
at 40 Gm. per cent and
above.
The circled
dots represent
hemoglobin
solutions
from
sickle-cell
trait
blood
(SA).
18
E
#{128}... ‘4
U)
0
(5
j)
2
>
i0#{149}
8-
6
4.
2-
08
2
6
HEMOGLOBIN
20
24
28
CONCENTRATION
32
36
1Gm/lOOm))
40
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
PHYSICAL
PROPERTIES
could
formation
OF RED
not
be
CELLS
857
I.
prevented
by continuing
manipulation
gelled
to a semisolid
state
on standing.
On redilution
bin returned
to a fluid
state.
There
was no apparent
stroma-free
from
300
showed
no
300, 600,
Washed
of the
ity,
change
in viscosity
disrupted
in
red
contrast
globin
to
intact
concentrations
the
hemolysis
moglobin
products,
of 30 Gm.
ml. of solution
which
varied
made
by
per
a concentration
by
NaCl
sonic
concentration
vibration
red cells
(Table
3).
of the hemolysis
products
only 15.6 Gm. per 100
of the intact
red cells
at
when
40 Cm.
of
per
was
cell
was
varied
stroma
adjusted
such
of the
experiments,
disrupted
to
contain-
water.
The
by differences
contrasted
with hemoglobin
from
33 to 47 Gm./100
ml.
sonic
vibration,
they were
too
cent,
In
solutions
in preparations
of 300, 600 and 1,200 mOsm./Kg.
cells was low and was not affected
concentrations
the
water
the hemogloon the viscosity
of
solutions
when
the concentration
of NaCl
600 mOsm.
Preparations
of washed
red
than
900 and 1,200 mOsm.
red cells were
disrupted
NaCl
ing
hemoglobin
greater
to
and
with
effect
viscosity
in tonicthe hemocells were
red
concentration
of cells. When
contained
concentrations
viscous
to measure
and
of hegelled
they
cent.
DIscUSSIoN
The
flow
because
to the
characteristics
of shear
rate.
In the microcirculation
than
the red cell. Bloch’2
normal
flow
red
or twisted
cells
within
diameter.
and
the
he
the
istic
shear
such
of
decrease
changes
during
evident
manner,
and
by
the
freezing
the
hemoglobin
The
viscosity
taraldehyde-treated
and
gelled
of the
were
thawing
and
fixed
cells
more
normal
Lindquist
The
of the
is also
plastic
fixed
diminured cell
character-
red
cells
may
remarkable
was
would
not
red
than
plasticity
shape
they
was
rigid
pass
and
through
should
behave
cells
because
by
could
vibration.
increased
cells
normal
or their
tested
more
to bered
In
this
model
not
be
hemolyzed
was
evidence
There
after
capacity
observing
or formaldehyde.
because
stroma
cells
rigid
was
or by sonic
the
hema-
the
viscosity
of decreasing
and
undergo
viscous
rigidity
glutaraldehyde
cells
elongated,
that
tubes
Fahraeus
and
rises
or larger
during
at a constant
normal
Dintenfass,’5
be
their
Increased
with
red
lost
that
fluidity
The
contrast
smaller
compressed,
cells
the
in
is
is independent
microcirculation.
by
have
rate
non-Newtonian
related
to viscosity.
increased
fluidity
virus.’5
shear
formulation
fluid.
treated
that
the
the
be
demonstrated
in capillary
confirm
property
mosaic
when
through
would
increasingly
chemically
water,
flow
cells
This
This
i.e.,
red
of increased
rises.
as tobacco
Newtonian
rigid
come
others’5
as occurs
not
as
rates.7”
constant,
in shape,
normal
rate
to the
in a more
and
rises,
described
vessels
may be
have
demonstrated
rates
with
viscosity
According
the
Linquist’4
shear
rate
of the
al.’3
changes
Studying
Mayer’#{176} did
gels
in
undergo
et
are
shear
remains
did not measure
shear
appears
to be correlated
in viscosity
when
the diameters
and Guest
may
Although
experiments14
blood
at lower
fluid that
milliseconds.
crit
Fahraeus
decreases
when
tion
whole
of
the viscosity
increases
viscosity
of a Newtonian
this
chemical
Newtonian.
a microfilter.
The
cells
it was
by
that
fixation.
The
glu-
fixed
red
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
858
HAM
cells
did
not
centrifugal
tube
that
indicated
change
in volume
but
showed
extreme
resistance
ET
AL.
to packing
by
force.
Normal
red cells, however,
packed
so well in the hematocrit
they were
translucent
to light
whereas
the fixed
cells were
not. This
that normal
cells were
so well adapted
to each
other
by their
chang-
ing shape
have
been
that
light
reported
was transmitted
by Chien
et al.’7
compared
to normal
cells.
The studies
of sickled
cells
sickle
cell disease
confirmed
for observations
were
hemolyzed
Sickled
showed
the
the
cells
mass.
Similar
hardened
by
observations
acetaldehyde
from patients
with sickle-cell
trait or homozygous
previous
observations8’9
and
serve
as controls
in hypertonic
by water,
cells
through
for red
media.
freezing
by
Sickled
and
following
cells
thawing
were
not fixed
and by sonic
characteristics:
resistance
since
they
vibration.
to
packing
by
centrifugal
force,
failure
to pass
through
microfilters
that
readily
permitted
the flow of unsickled
forms
as described
by Jandl
et al.18 and an extreme
increase
in viscosity
that was greater
than
observed
for cells fixed
in glutaraldehyde.
It was concluded
that
sickled
cells had lost fluidity
and were
therefore
rigid,
due
to tactoid
gel formation.
hemoglobin
that
plasticity
NaCl!Kg.
to result
of the
hemoglobin.8
solutions
Hypertonicity
solutions
or the
tonicity
by the
nor
were
there
therefore,
loss of
foundation
hemoglobin
Stroma-free
addition
shifts
in
of
40
Gm.!
did
not
change
viscosity
of washed
of urea
water
did not
altering
100
ml.,
or
above,
the
viscosity
of
red cell stroma.
alter
the
showed
stroma-free
Increasing
the physical
properties
MCHC.
There
was
all samples
of red cells
studied
when
exposed
to hypertonicity
showed
from
of water.
This
loss of deformability
directly
from
the high
concentration
of cells
evidence,
increased
rigidity
with
600 to 1,200 mOsm.
of
or increased
of hemoglobin
rigidity
within
appeared
the cell.
Under
these
conditions,
loss of fluidity
was probably
due to a change
in the
physical
properties
of the hemoglobin,
such
as gel formation.
This mechanism
for increased
rigidity
has been
suggested
by Erslev
and Atwater.19
Rand
and
Lacombe2#{176} have
centrations
of
observed
hypertonic
media
and related
these
it was
observed
media
viscosity
occurred
of hemoglobin.
dogs,
at
pH
normal
humans,
These
results
stein.2’
These
authors
solutions
from
the
with
reported
sickle
in
results
were
They
cell
disease
is
not
the
increase
no
that
or
an
These
viscosity
media.
from
sickle
of Perillie
in viscosity
authors
blood
homozygous
forms
incon-
resistance
oxygenated
sickled
in the
When
increased
filtration,
for
and
concentration
their
work,
incubated
in
MCHC.
with
an increase
in hypertonic
clear.
con-
observed
observations
in
increase
found
increasing
They
decreased
trait
with
an
but
trait.
viscosity
identical
sickle
at variance
chloride,
in
with
hemoglobin
In extending
in red
cells
it occurred
in viscosity,
changes
are
sickle-cell
homozygous
differences
increase
rose
NaCl.
increased
viscosity.
occurred
crenation,
patients
of sodium
from
the
of blood
hypertonic
a change
with
morphologic
disease.
or those
8 without
media,
and
viscosity
and
produced
crenation
and
changes
to the increased
that
crenation
per
se
isotonic
In hypertonic
the
dextrose
creased
centration
to packing
that
mannitol,
in
and
cell
Ep-
hypertonic
of normal
only
The
explanation
have
proposed
cells
in cells
for
that
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
PHYSICAL
PROPERTIES
OF RED
in vitro
hypertonicity
medulla
microcirculation
and
disease.2528
in homozygous
Whitten29
sickle
renal
CELLS
resulted
859
I.
in sickling
vasa recta2224
and thus
lead
has
cell
and
that
hypertonicity
in vivo
would
result
in sickling
in these
to the renal
disease
in homozygous
studied
disease
the failure
in response
of renal
to the
in the
areas
of the
sickle
cell
concentrating
protein
content
capacity
of the
diet. He believes
the defect
is a failure
of the vasa recta
and does not confirm
the hypotheses
of Perille
and Epstein?’
It is possible,
however,
that
new
information
submitted
here
may
explain
the
abnormal
function
of the
vasa
recta as outlined
in the following
steps,
to form a hypothesis.
It has
increased
been
demonstrated
that hypertonicity
of sodium
viscosity
of all cells but did not cause
sickling
chloride
resulted
in
of oxygenated
blood.
increased
viscosity
could
result
in decreased
flow in the vasa
recta
for
all bloods,
coincident
with
rising
concentration
of sodium
chloride.
The
decreased
rate of flow could
lead to lowered
O2
and pH and these
in turn may
result
in sickling
in both
homozygous
and heterozygous
sickle
cell disease.3#{176}
Sickling
increases
viscosity
significantly
and could
affect
the vascular
bed of
This
the medulla,
ited kidney
oxygen
accounting
for thrombosis,
function
with hyposthenuria.2528
tension
is variable3032
and
papillary
The
sickling
necrosis,
hematuria
degree
of sickling
takes
a finite
and limat lowered
period.31’32
The
rate
of circulation
through
the kidney
may
affect
the degree
of sickling
and
the
degree
of obstruction
in the microcirculation,
and therefore
produce
a variable
degree
of renal
involvement.
Levitt
et al.28 could
reverse
the renal
defect
of hyposthenuria
in homozygous
sickle
cell disease
by multiple
transfusions
of
normal
blood
in children
up to approximately
5 years
of age but not in older
children.
Physiologically,
of the vasa recta when
The red
than those
in viscosity
slow
the
the
this
normal
cells in sickle-cell
of the homozygous
which
results
blood
critical
flow
level
with normal
to the kidney.
functioning
trait
are
less susceptible
to the sickling
disease.
In sickle-cell
trait, however,
the
from
hypertonicity
at a normal
hematocrit
to such
for
reversibility
is consistent
cells are made
available
a degree
sickling.
that
deoxygenation
Accordingly,
the
and
kidney
tion in which
the sickling
process
is enhanced,
and
with
heterozygous
sickle
cell disease
are vulnerable
as are those with the homozygous
disease.
lowered
presents
process
increase
may
pH
a special
reach
situa-
as a consequence,
patients
to renal
complications,
SUMMARY
Changes
fixation,
and
in the
the
physical
sickling
by a combination
These
indicated
Newtonian
resistance
mechanical
sodium
relationship
resulted
by
a rise
to
a loss
in
of
packing
all
of these
by
cells
of
have
physical
an
or
cell
these
increased
properties
increased
suspensions
by
chemical
sodium
chloride
rigidity
and
suspensions
force
of
produced
hypertonic
sickling.
of cell
centrifugal
results
were
in
and
filtration
The
cells
suspension
defor.mabiity
viscosity
diminished
trauma.
chloride
of red
by
of hypertonicity
in
flow,
properties
process,
and
abnormal
show
viscosity
behavior
and
cells
hemolysates,
through
studies
to the
of
as
more
microfilters,
susceptibility
that
are
in
more
of cells
to
hypertonic
rigid.
in the
The
micro-
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
860
HAM
circulation
including
is discussed
for normal
the effect
on the kidney.
subjects
IN
SUMMARIO
and
those
with
sickle
cell
fiu.xo newtonian,
resistentia
contra
trauma
pro
mechanic.
de natrium
iste
inter
Le
omne
le cellulas
proprietates
physic
discutite
pro
le effecto
super
un
paccage
per
fortias
de
suspensiones
centrifuge,
e
con
subjectos
morbo
a
rigiditate
cellular,
a
del
Ufl
transverso
susceptibilitate
un
de iste studios
monstra
que in
ha un augmentate
viscositate
e es plus
e le comportamento
del cellulas
in le
e pro
disease,
per fixation
de natrium,
cellular
anormal
hypertonic
resultatos
normal
subjectos
filtration
reducite
AL.
INTERLINGUA
Alterationes
in
le proprietates
physic
del erythrocytos
esseva
producite
chimic,
per le processo
falcfficatori,
per suspension
in hypertonic
chloruro
e per un combination
de hypertonicitate
e falciformation.
Le proceduras
resultava
in un perdita
de deformabilitate
o un augmentate
cellulas,
manifeste
in un augmentate
viscositate
de suspensiones
e hemolysatos
intensificate
microfiltros,
ET
chloruro
rigide.
Le
relation
microcirculation
cellulas
falciforme,
es
incluse
le renes.
ACKNOWLEDGMENTS
is expressed
to Dr.
of red cells; to Dr. Frederick
of Harvard
Medical
School
Carl F. Hinz for suggesting
the
E. Wheelock
for help in microscopy;
Appreciation
fixatives
E.
Wells
determining
and
the
Peter
Bent
Brigham
of chemical
and to Dr. Roe
use
Hospital
for
help
in
viscosities.
REFERENCES
1.
T. H.,
Ham,
and
Sayre,
R. W.,
Dunn,
R. F.,
J. R.: Physical
properties
of
red cells as related
to effects
in vivo. 2.
Effect of thermal
treatment
on rigidity
of
red cells, stroma
and the sickle cell. Blood,
Murphy,
32:862,
2.
1968.
Carlson,
cal
J. and Ham,
D.
properties
red
of
T. H.: Physirelated
to
cells
as
in vivo.
3. Effect
of thermal
treatment in survival
of red cells in the dog role
of the spleen.
Blood, 32:872,
1968.
effects
A. K., Lorenze,
P. B., Parpart,
E., Gregg,
J. R., and Chase,
A. M.: The
osmotic
resistence
(fragility)
of human
red
cells. J. Clin. Invest.
26:636,
1947.
4. Page,
L. B., and Culver,
P. J.: A syllabus of laboratory
examination
in clinical
di3.
Parpart,
agnosis,
sity
Cambridge,
Press,
5.
and
ties
Mass.,
Harvard
Univer-
influence
of pH
proper-
1960.
J.
Murphy,
The
some
R.:
on
temperature
of normal
erythrocytes
from patients
J. Lab. Clin.
physical
and
erythrocytes
with
hereditary
spherocytosis.
Med. 69:758,
1967.
6. Wells,
R. E., Denton,
R. and Merrill,
E. W.,
Jr.:
Measurement
of viscosity
of
biologic
fluids by cone plate viscometer.
J.
Lab.
7.
Influence
Clin.
Med.
Wells,
R.
of
flow
57:646,
E.,
Jr.
1961.
and
properties
Merrill,
of
blood
E.
W.:
upon
viscosity-Hematocrit
relationships.
J. Clin.
Invest.
41:1591,
1962.
8. Harris,
J. W.: Studies
on the destruction of red blood
cells:
VIII.
Molecular
orientation
in
sickle-cell
hemoglobin
solu-
tions.
Proc.
Soc. Exper.
Biol. Med.
75:197,
1950.
9. Ham, T. H., and Castle,
W. B.: Relation
of increased
hypotomc
fragility
and
of
erythrostasis
to the mechanism
of hemolysis
in
certain
anemias.
Trans.
55:127,
1940.
10. Shen,
S. C., Castle,
ing,
E. M.: Experimental
servations
on increased
Ass.
W.
Amer.
B. and
Phys.
Flem-
and clinical
obmechanical
fragility
Science
100:387,
1944.
11. Wilkinson,
W.
L.:
Non-Newtonian
fluids.
Fluid
mechanics,
mixing
and
heat
transfer,
New York,
Pergamon
Press,
1960,
p. 138.
12. Bloch,
E. H.: A quantitative
study
of
the hemodynamics
in the living
microvascular system.
Amer.
J. Anat.
110:12.5,
1962.
13. Guest,
M. M., Bond,
T. P., Cooper,
R. G., Derrick,
J. R.: Red blood
cells:
Change
in shape
in capillaries,
Science
142:
1319, 1963.
14. Fahraeus,
R. and Lindquist,
T.: The
viscosity
of the blood
in narrow
capillary
tubes.
Amer. J. Physiol.
96:562,
1931.
of
erythrocytes.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
PHYSICAL
PROPEIITIES
OF RED
CELLS
861
I.
15. Dintenfass,
L.: Considerations
of the
effect
of the internal
viscosity
of red cells
and
its effect
on
the viscosity
of whole
blood.
Angiology
13:333,
1962.
16. Mayer,
C. A.: Anomalous
viscosity
of
human
blood.
Amer.
J. Physiol.
208:1267,
1965.
17. Chien,
S., Usami,
S., Dellenback,
R.
J. and Gregersen,
M. J.: Blood viscosity:
Influence
of
157:827,
erythrocyte
deformation.
Science
1967.
18. Jandl,
J. H., Simmons,
R. L. and
Castle,
W. B.: Red cell filtration
and the
pathogenesis
of certain
hemolytic
anemias.
Blood 18:133,
1961.
19. Erslev,
A. J. and Atwater,
J.: Effect
of
mean
tration
corpuscular
on
hemoglobin
J. Lab.
viscosity.
concen-
Clin.
Med.
62:
401, 1963.
20. Rand,
P. W. and Lacombe,
E.: Hemodilution,
tonicity,
and blood
viscosity.
J.
Clin. Invest.
43:2214,
1964.
21. Perillie,
P. E. and Epstein,
F. H.:
Sickling
phenomenon
produced
by hypertonic
solutions:
A possible
explanation
for
the
hyposthenuria
of sicklemia.
J. Clin. Invest.
22.
tration
Med.
23.
B.:
kidney
42:570,
1963.
Gottschalk,
and
C.
dilution
36:670,
concenAmer.
J.
1964.
Pappenheimer,
Hematocrit
ratio
J. R. and
within the
its significance
and
ics. Amer.
W.:
Osmotic
the urine.
of
J.
Physiol.
185:377,
for
Kinter,
W.
mammalian
renal
1956.
dynam-
24.
Thurau,
J. Med.
K.:
Renal
hemodynamics.
36:698,
1964.
25. Keitel,
H.
G.,
Thompson,
D. and
Itano,
H. A.: Hyposthenuria
in sickle
cell
anemia:
A reversible
renal
defect.
J. Clin.
Invest.
35:998,
1956.
26. Mostofi,
F. K., Bruegge,
C. F. and
Diggs,
L. W.: Lesions
in kidneys
removed
for
unilateral
hematuria
in sickle
cell disease. Arch. Path. 63:336,
1957.
27. Schlitt,
L. E. and Keitel,
H. C.: Renal
manifestations
of sickle
cell disease:
A review. Amer. J. Med. Sci. 239:773,
1960.
28. Levitt,
M. F., Hauser,
A. D., Levy,
M. S. and Polimeros,
D.: The renal concentrating
defect
in sickle
cell disease.
Amer.
J. Med. 29:611,
1960.
29. Whitten,
C. F.: Effect
of dietary
protein
on the
renal
concentrating
process.
Amer. J. Dis. Children
115:262,
1968.
30. Lange,
R. D., Minnich,
V., Moore,
C. V.: Effect
of oxygen
tension
and of pH
on the sickling
and mechanical
fragility
of
erythrocytes
from
patients
with sickle
cell
anemia
and the sickle
cell trait.
J. Lab. Clin.
Med. 37:789,
1951.
31. Jensen,
W. N., Rucknagel,
D. L. and
Taylor,
W. J.: In vivo
study
of the
sickle
cell phenomenon.
J. Lab. Clin. Med. 56:
854, 1960.
32. Pearson,
H. A., Schieber,
C. L., Krovets, J. J., Bartley,
T. D., and David,
J. K.:
Sickle
cell anemia
associated
with tetralogy
of fallot.
New Eng. J. Med. 273:1079,
1965.
Amer.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1968 32: 847-861
Physical Properties of Red Cells as Related to Effects in Vivo. I. Increased
Rigidity of Erythrocytes as Measured by Viscosity of Cells Altered by
Chemical Fixation, Sickling and Hypertonicity
THOMAS HALE HAM, REBECCA F. DUNN, RICHARD W. SAYRE and JOHN R. MURPHY
Updated information and services can be found at:
http://www.bloodjournal.org/content/32/6/847.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of
Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.