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A Simple
Laboratory
Alternative
Sickled
By Margaret
Irreversibly
sickled
hallmark
eral
blood
gous
SS
ISC
cells
of sickle
smears
varying
difficulty
cells.
For
this
alternative
than
smears.
Because
surement
A
of ISC
in standardizing
the
we
have
the
microscopic
ISC
are
on
cell
based
of
dehydrated
an
However,
assessment
less
blood
of the
a mea-
clinical
implica-
categorization
ently
subject
have
sought
quantification
but rather
reproducibly
of cell morphology
and is thus inherto variation.
With
this perspective,
we
to develop
an alternative
method
for
of ISC that is based
not on morphology,
on a property
that
could
be measured
and objectively.
Such
a property
is the
low ion and
water
mean
and
content
ity.3 In the experiments
relationship
between
and
the
from
The
of
more
high
high
heparin
For
drawn
from
or EDTA,
ISC
and
counts,
for 30 mm
and
M
phosphate
buffer,
wet
slide
length
was
presence
of
enced
Whole
and
Blood,
spiculated,
All
individual
cell
elongation
and
5
cells
in
width,
performed
counts
(M.R.C.)
deformability,
cells
Vol. 60, No. 3 (September),
fluid
were
cells
samples
performed
to minimize
defined
was
blood
low
also
observer
the
1982
stress
Cells
water
Parallel
these
substitute
for
the
measure-
high
MCHC
ISC
counts.
of ISC,
These
were
findings
sensitive
could
microscopic
that
of ISC,
measurements,
the
pro-
parameter
and
properties
on a
of morpho-
ektacytometric
with
of
between
percentage
another
content
either
was measured
laser
obtained
to
for
(PVP,
The
to
be used
counting
avg
25 zI whole
as an
of ISC.
the
from
experi-
had
shear
stress.
proportion
w/v)
of high
gradient
Tacoma,
The
micropipets
introducing
(-.1.140
first, a small
(-.10
g/mI),
then
a small
Stractan
at a density
space,
into
the
tube.
of the tube,
St.
Louis,
The
entire
which
Mo.)
Heights,
sample,
and
two
the
I . I 056
of this
packing
layer.
of the
Francisco,
Medical
Center.
Supported
dense
is publication
tology
Research
for
layer,
a standard
Co.,
Stractan
the
For
each
During
the
spin
spin,
Laboratory
of plasma
the
cells
cushion
tube
Lyon
tubes
that
extensive
The
collected
and
University
Memorial
that
remained
reduced
sealant.)
were
cells
Medicine
of Medicine,
Need-
20 mm.
2 fractions;
and
dry
Medical,
gradient
of the
air
drawn
to the
to fit into
of Hematology/Oncology.
Oakland,
close
in triplicate.
end
the Bruce
another
(Sherwood
spun
by
Stractan
20 MI of
was
Equipment
to provide
against
of
and
in part
This
drawn
cut
were
the
Department
Department
then
a three-layer
off
Department
Paper
l00-d
blood
Critoseal
Stractan
cells
Institute,
San
At
g/mI
Regis
introducing
were
leaving
(The
dense
the
Research
was
tubes
broken
a simple
(St.
dense
about
oxygenated
(International
of Stractan.
using
II
After
with
prepared
penetrated
on top
g/mI.
were
and
measured
the
N.Y.)
gradients
scored,
Mo.).
Corning,
they
layers
was
Stractan
of well
1). The
Louis,
Works,
gradient
eliminated,
St.
Glass
in which
removed,
Co.,
of 22 cp to provide
tl) cushion
of very
air space
and then
Mass.),
were
(290
in
5 l
centrifuge
of isotonic
prepared
was sealed
(Fig.
microhematocrit
were
were
of 1.1056
I 0-I
that
values
gradients
(Corning
approximately
end
cells
from
This
(DI)
polyvinyl-pyrolidone
Chemical
density
Wash.).
of
a viscosity
prepared
Corning
nia,
whose
Sigma
(4%
DI
in 3 ml
solutions
wt 360,000;
index
ellipticity.
suspended
oxygenated
mol
density
Co.,
blood
described.4
a deformability
cellular
solutions
appropriate
2-step
as previously
provides
average
well
PVP
The
the ektacytometer
viscometer
is proportional
ratory.
sample
excluded
using
diffraction
to
pronounced
Cancer
of Califor-
Research
Children
Labo-
‘s Hospital
Calif
by USPHS
number
Grants
33from
Laboratory,
HL
20985
and
AM
the MacMillan-Cargill
University
of California,
26263.
Hema-
San
Francis-
co.
Submitted
Address
Research
February
reprint
16. 1982:
requests
accepted
to Margaret
Institute.
University
by Grune
cc Stratton,
of California,
April
R. Clark,
San
23. 1982.
M-1 282,
Francisco,
Cancer
Calif
94143.
variation.
of cell
the
deformability.
physical
objective
in a
samples,
by a single
of
lSC.
and
samples
in a microhematocrit
correlation
cells
correlated
that
special
hr.
in 0.05
inadequate
were
5-6
counted
few
as the extent
shear
strongly
suggest
air spaces
into
exposed
with
In a very
indicated
these
were
at a standard
the
blood
in crisis,
within
blood
smooth
cells
and
not
microscopy.
as ISC.
sickled
reflects
ham
sickle
cell
observed
ml of 3% glutaraldehyde
or
cell
From
patients,
Five-hundred
counted
the
that either
cell dencould
be usefully
counts.
phase-contrast
fixation,
I5C
SC
were
in 0.5
7.4.
their
were
before
consideration.
fixed
under
cells
here, we studied
cell deformability
50 zI of whole
pH
twice
contours,
oxygenation
35 55
identified
blood
spun
a strong
density
of
of whole
gradient.
showed
of high
ments
METHODS
measurements
then
preparation
angular
AND
approximately
air
the
than
40 patients
with
degree
of correlation
MATERIALS
was
gives
density
among
these parameters
suggests
sity or whole
cell deformability
employed
as an alternative
to ISC
Blood
which
hemoglobin
concentration
as well as low cell deformabilpresented
ISC counts,
proportion
samples
disease.
of ISC,
corpuscular
density,”2
portion
Analysis
density
mosmole/kg),
tions of these cells has been hampered
by the fact that
they
cannot
be reproducibly
quantified.
The microscopic
counting
of ISC requires
individual,
subjective
a high
(MCHC)
approach.
H. Lubin
attractive
in the underof sickle
cell
sickled
cells
QUESTION
standing
of the pathophysiology
disease
has been the role of irreversibly
(ISC).
on
cells.
seemed
an
be
and Bertram
2-step
logically
develop
cells
dense
density
disease.
would
alternative
centrifuge,
for
of these
to
that
H. Embury,
simple.
complicated
quantification
counting
LONG-STANDING
the
been
attempted
a
a role
of
of quantification
be
homozy-
suggested
have
Stephen
in periph-
different
has
role
to
number
among
(ISC)
Mohandas,
considered
manifestations
the
reason,
Narla
their
variation
clinical
method
variable
are
yet
greatly
This
to determine
by the
(ISC)
disease,
varies
patients.
the
in
Efforts
cell
R. Clark,
Cell
to Irreversibly
Counts
© 1982
orientation
I 25 dyne/sq
cm,
Inc.
0006-4971/82/6003-0017$O1.OO/O
659
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
CLARK
660
fractions
0.1%
were
fraction
X-I0O
was
ments
all
then
in cyanomethemoglobin
to ensure
calculated
of hemoglobin
cells
in
resolution,
a
sample
for a small
hemoglobin
content
of the
minor
mature
proportion
determine
the
had
gradient
proportion
of these
cells
and
of the
total
proportion
lysis;
the
fraction.
the
reagent
the
from
in each
multilayer
except
that
dissolved
Triton
percentage
spectrophotometric
This
same
showed
cells.
cells
generally
was
the
cells
the
was
that
this
was
the
mean
more
cells
use
virtually
of reagent,
measurements
that
content.
Because
young
population,
of dense
assumes
hemoglobin
analysis
the use ofcell
in each
measure-
calculation
of young
because
containing
of cells
than
those
cells
High
reproducibility
true,
determinations
90%
constituted
cell
I 3.3%
a
layer
of
to
restrained
l,Table
dense
had
a
portion
density
gave
a value
Preliminary,
1.1056
the vast
g/ml
was
usually
was dissolved
proportion
greater
on
a sickle
of 28.0
±
multilayer
allowed
majority
than
cell
for
gradient
analyses
of
virtually
normal
blood
in 2 ml
The
I 2 replicate
that
the
AL.
From these
cells,
i.e.,
g/mI.
high;
sample
I .3 (SD)
from
1.1056
was
passage
ofcells
dissolved
in 4 ml.
of “dense”
cell determination
performed
cells.
to
that
ISC
dense
equivalent
The
the upper
fraction
we calculated
the
of the dense
of
of hemoglobin
counts.
and
ET
contained
percentage
showed
all
of
that
ISC,
samples
a
but
(Fig.
I).
U
ii ii
MN
cont
FHVH
SS
SS
Fig. 1 .
(A)
Microgradients
used to determine
the proportion
of high density
cells. Duplicate
samples
from one normal
subject
(MN.)
and two sisters (F.H. and V.H.) with homozygous
SS disease
are shown.
The dark band between
the first Stractan
layer (p - 1 .1056
g/mI)
and the plasma represents
the low density fraction.
and all the cells below that cell/Stractan
interface
represent
the high density
fraction.
See Table
1 for the proportions
of dense
cells. ISC counts.
and deformabilities
for these
samples.
(B) High resolution
discontinuous
Stractan
g/ml
gradients
increments.
of samples
shown
in A.
The
densities
of the
Stractan
layers
ranged
from
1 .070
to
1 .1 1 5 g/ml.
in approximately
0.004
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
ALTERNATIVE
661
TO ISC COUNTS
Table
1 . Prope rties
of B lood Sam pIes in Fig
.
1
Percent
MCHC
Dense
Percent
Cells
SC
Dl2,,
mability
and
samples
from
ISC
counts
subjects
was
who
SS-F.H.
36.6
36.4
12.0
57.4
(Fig. 3). As with the density
of SC samples
was reduced
appreciable
numbers
of ISC,
SS-V.H.
36.6
22.3
6.8
71.0
consistent
(g/dI)
Normalcontrol-
MN.
33.7
3.8
(%
Control)
100.0
-
with
morphological
cell
also
had
observed
for
homozygous
blood
SS disease
assay,
the deformability
even in the absence
of
and this finding
is also
dehydration
unaccompanied
by
changes.
RESULTS
We
found
counts
and
a very
the
percent
had
homozygous
finding
is consistent
the vast
density
strong
majority
regions
correlation
of dense
sickle
with
cells
between
for patients
who
(Fig.
2).
observation
This
that
cell disease
our previous
of ISC are concentrated
during
density
gradient
tion.2
with
It should
densities
than
some
the percentage
of ISC, suggesting
cells that were not morphologically
DISCUSSION
ISC
In these
studies
we
method
for quantitating
abnormal
water
content
in the high
centrifuga-
The
be noted
that the percentage
of cells
greater
than
1.1056
g/ml
was higher
dehydration
identifiable
As expected
the major
bility,3
from our
determinant
a strong
tively.
of
60
previous
finding
that MCHC
of reduced
ISC deforma-
correlation
between
whole
cell
bouyant
both
A strong
of these
and
decreases
of which
correlation
was
cells
increases
their
can
counts
and both the proportion
cells and the cell deformability.
whole
be measured
found
cell
objec-
between
of abnormally
This finding
ISC
dense
suggests
that measurements
either
of deformability
or density
distribution
could be used as an objective
alternative
to
ISC counts.
Because
of the fact that it uses generally
available
laboratory
equipment,
the density
assay
should
be especially
useful.
It should
defor-
dehydration
density”2
deformability,3
as ISC. Interestingly,
the ISC versus
density
correlation did not hold for blood samples
from patients
with
SC disease,
in which there were substantial
numbers
of
high density
cells, but very few ISC.
was
extraordinary
their
have developed
and tested
a
ISC on the basis
of their
rather
than cell morphology.
be noted
that
neither
density
nor deforma-
bility
measurement
directly
measure
ISC, which
defined
morphologically.
This
fact is exemplified
our measurements
of blood samples
from subjects
-
are
by
who
A
lOOr
50-
A
(1)
801
-J
-I
w
40-
0
A
U
A
A
A
-
6000
0
-J
0
Ui
0
0
I-
A
-
z
%
z
A
>-
Ui
(.1) 30
0
4
AAA
A
20
40-
0
0
0
a:
00
0
A
20-
0
0
I0
0
0
lO
20
30
40
2.
Percentage
of
dense
cells
(cells
that
penetrated
a
Stractan
layer of 1 .1056 g/ml density)
versus percentage
of ISC in
45 blood samples
from 37 SS and SC patients.
For the SS samples
(open symbols).
a correlation
coefficient
of 0.92 was obtained
from
a linear
regression
analysis.
The SC samples
(solid
symbols)
contained
moderate
numbers
of dense
cells
but
very
few
ISC.
I
I
I
I
20
30
40
50
%ISC
50
%ISC
Fig.
I
10
Fig. 3.
Deformability
index
(Dl) at 125 dynes/sq
cm shear
stress versus percentage
of ISC for 49 blood samples
from 40 SS
and SC patients.
Dl is given as the percentage
of the instrument
reading
for normal
control
cells at the same shear stress.
For the
SS samples
(open symbols)
a correlation
coefficient
of - 0.96 was
found. As for the data in Fig. 2, the SC samples
(closed
symbols)
showed
a disproportionate
decrease
in Dl for their very low ISC
counts.
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
CLARK
662
had the HbSC
disease,
which
showed
vations
in cell density
and impairment
substantial
eleof cell deforma-
involved
in vasoocclusion
where
the deformability
major
deformability
phology
for
cell disease.
In addition,
our findings
suggest
that
comparative
studies
of SC and 55 patients
and of their
red cells might
elucidate
the relationship
between
cell
dehydration
and abnormal
morphology
in the homozy-
be more
important
than cell morpathologic
processes.
Because
of
the extreme
dependence
of sickling
kinetics
and
mer
formation
on hemoglobin
concentration,6
high MCHC,
dense
cells might
be disproportionately
polywith
of blood
microcirculation,
red cells is the
bility
in the absence
of appreciable
numbers
of ISC.
Thus,
clinical
use of the density
assay
requires
accurate prior diagnosis
of the type of hemoglobinopathy
present.
However,
it may be that cell water content
and
may
certain
determinant
of the
of individual
ET AL.
flow.7’8
Correlation
of the
results
of the density
assay with some kind of clinical
severity
evaluation
should
be useful
in determining
the
rheologic
implications
of cellular
dehydration
in sickle
gous
disease.
REFERENCES
I
.
Glader
Nathan
sickled
2.
sition
BE,
DG:
cells
MR.
AlP
genated
4.
factors
Clark
Muller-Soyano
A, Platt
and
composition
cation
Br i Haematol
Unger
and
51:1169-1178,
3.
SE,
reserve
in vivo.
Clark
and
Lux
Energy
RC,
lipid
40:527-532,
Shohet
content
OS,
SB:
RD.
cation
deoxy-hemoglobin
6.
sickled
compo-
cells.
Blood
4868,
7.
1978
MR.
Mohandas
N,
irreversibly
sickled
cells.
Mohandas
N, Clark
regulating
MR.
Shohet
i Clin
Jacobs
erythrocyte
SB:
Invest
MS.
deformability.
Deformability
65:189-196,
Shohet
J Clin
Corash
SB:
Piomelli
5,
Chen
HC,
Seaman
C,
Hofrichter
Mechanics
of
GL
8.
Gross
I 980
PL:
to age
1974
PD,
Behavior
Cokelet,
Hi
and
Blood
Driessen
Sch#{246}nbein H:
E:
i, Ross
according
84:147-151,
Eaton
WA:
Proc
on a simplified
Kinetics
NatI
and
Acad
Sci
density
mechanism
USA
of
71:4864-
1974
1980
66:563-
Med
S gelation.
La Celle
in
Analysis
Invest
J LabClin
of oxy-
velocity
LM,
of erythrocytes
gradient.
573, 1980
5.
Separation
1978
Monovalent
of irreversibly
Propper
of irreversibly
CK,
Effect
in capillaries
of abnormal
Meiselman,
Flow,
Haest
of
DE
New
CWM,
reduced
of rat
York,
erythrocytes
Brooks
AR
Heidtmann
red
mesentery.
cell
in capillaries,
(eds):
Liss,
Erythrocyte
I 980,
pp 195-209
H, Kamp
D, Schmid-
deformability
Pflugers
Arch
on
388:75-78,
flow
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
1982 60: 659-662
A simple laboratory alternative to irreversibly sickled cell (ISC) counts
MR Clark, N Mohandas, SH Embury and BH Lubin
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