The
Measurement
of Lymphocyte
Particle
Deformability
George
By
We
have
determined
deformability
the
and
influence
suspending
and
B. Segel.
Giles
of reference
buffer
tonicity
however.
was
lymphocytocrit/lymphocyte
lymphocyte
weight
difference
between
21 8 cu
lymphocyte
the
of lymphocyte
T
HE
when
volume
of 21 0 cu tm
ELECTRONIC
particle
volume
by the
due to
factor)
analyzer
To ensure
were
Biophysics.
cine
Dentistry.
23355.
and
Research
of
Rochester,
in part
by
HL-18208.
Jimmy”
Pediatrics,
N.
USPHS
by
Fund,
filter
of
of
and
(no.
of
Grants
University
by the
Ruth
of
CA-l2790.
Goldberg
were
Address
Rochester
August
reprint
School
29,
1980;
requests
accepted
to George
of Medicine.
Elmwood
Avenue,
Rochester,
J 981 by Grune
& Stratton,
0006-4971/81/5705-00l3$Ol.OO/O
894
l0
GIBCO,
cells/mi
and
volume
of twice
solution
contain-
salt
This
tightly
eluted
from
Ashland,
the
in
Forty
50-mI
a
column
in this
suspension
was
from a leukopheresis
Mass.).
packed
prepared
cell
prepared
with
way
milliliters
syringe.
20
ml
contained
The
portions
fewer
of
than
ofLymphocyte
and Lymphocyte
of
in
then
cell
6
microliters
Foun-
Instruments,
suspensions
unlabeled
sucrose
60 tl
the
were
of the total
was
N KOH.
The excess supernate
height.
to
was
measure-
g for 6 mm
cell
The
and
added
of
per
suspension
packed
scintillation
plasma
was added
at 8000
the
column
Ill.)
AB
microcuries
this
of the supernatant
Grove,
20%
8 replicate
sedimented
and
supernatant
Downers
of
to provide
centrifuge
at the juncture
of
Three
tubes
as a percentage
severed
plus
cells/mI.
Precisely
microhematocrit
cell
in TC-l99
l0
I .66 mmole
microhematocrit
Blood
x
suspension.
into
These
a
suspended
of
containing
the
ments.
Medi-
were
a concentration
Volume
by
Count
volume
tubes
pellet.
Instagel
fluid
were
Twenty
(Packard
with
80 sl of
0.625
Submitted
(C)
a balanced
Inc.,
were
Lymphocytes
Measurement
Lymphocytocrit
dation.
601
2 x
I .5% monocytes.
ilL-
Rochester
Estrin
cell concentrates
an equal
lymphocytes/mI.
Fenwal,
material
measured
Research
cell-rich
described.8
199 (TC-199),
of
at 37#{176}C
with
reagent,
l0
4C2450;
filter
TC-l99.
Radiation
School
the white
medium
a nylon fiber column
lymphocytes
Y.
the
and
Medicine,
of Rochester
volume
mononuclear
as previously
at a concentration
lymphocyte
passed through
of the true
lymphocyte
on an electronic
particle
University
culture
for 30 mm
concentration
pipetted
Departments
true
ing iron filings (Technicon
Inst. Corp., Tarrytown,
N.Y.).
The
mononuclear
cells were then separated on a step
and the
mononuclear
cell layer was harvested, washed, and resuspended at a
ml
Supported
N.Y.)
the
donors
of monocytes,
in tissue
Island,
‘4C-sucrose
the
the
METHODS
from
of healthy
a low proportion
suspended
Lymphocytes
and
Since
AND
prepared
residues
concentrated
at
and
were
plateletpheresis
analyzer.
From
aperture.
of Lymphocytes
Lymphocytes
incubated
measured
lymphocyte
volume
by two
independent
methods
to allow calculation
of a shape or deformability factor,f,
for lymphocytes.
Knowledge
of this shape
Biology
sizing
MATERIALS
Grand
by assessing
the effect of standard
particle
deformability and suspending
buffer
tonicity
on the electronic
measurement
of lymphocyte
volume.
Further,
we have
volume
the
Preparation
for use in a clinical
laboratory
standardization
has been
estabblood
red cells,
white
cells,
and
determination
measurements
traverses
has
of the same nucleated
cells. For example,
the reported
lymphocyte
volume
varies
from
180 to 280 cu
We have explored
the reasons
for the marked
variation
allows
from
A. Lichtman
as
platelets,
but for sizing
only human
red cells.
When
such instruments
have been used in research
laboratories, there
has been a marked
variation
in the volume
factor
volume
Marshall
equals the Vs/f,.
the red cells with a shape factor near 1.0
were sized appropriately
by this method.
In contrast.
the
lymphocyte
shape factor was 1 .38; thus. the true lymphocyte volume
was 289 cu tm/1.38
or 210 cu tim. The
tonicity
of the suspending
solution
also influenced
the
measurement
of particle volume when osmotically
inactive
standard
particles
(e.g.. latex spherules)
were used as a
reference.
Whereas
the true lymphocyte
volume was 210
cu ,m at 286 mosmole/liter.
it was 194 cu m
at 330 and
229
cu m at 250 mosmole/liter.
The standard
Counting
solution.
Isoton. is hyperosmolar
(330 mosmole/liter)
and
causes
an 8% shrinkage
of osmotically
active cells.
the
become
a standard
tool
in many
biomedical
research
laboratories.
Many
of these instruments
were
designed
principally
setting.
Therefore,
lished
for counting
and
Tonicity
it
was
(shape
Solution
particle
measured
deformability
Counting
of Reference
on
count
and 203 cu m
by wet
and density
(mean
21 0 cu im). The
the electronic
volume
( V.) of 289 cu
Mm and true
influence
sm
Importance
R. Cokelet,
measurement
of lymphocyte
volume
by an electronic
particle
volume
analyzer.
When the volume
analyzer
was
standardized
with latex spherules
having a shape factor
(f.) of 1 .5. red cell volume was 96 cu m and lymphocyte
volume was 289 cu m. The red cell volume corresponded
closely to the true red cell volume;
the true lymphocyte
volume.
Volume:
January
B. Segel.
Department
N. Y. 14642.
Inc.
8. 1981.
M.D.
,
University
of Pediatrics.
Box
of
777,
and
the
pellet
containing
dispersed
thawed
incubation
0.4
and
and
pushed
ml
distilled
allowed
digested
at
was blotted
was
the
water.
to freeze
at
with
l
37#{176}C,the
from
from
100
digest
the end of the cell
severed
The
-
of
was
tube
pellet
into
was
pellet,
a tube
thoroughly
20#{176}C
for 45 mm. It was then
2.5
N
KOH.
throughly
Blood,
Vol. 57.
mixed
After
and
No. 5 (May),
30-mm
100
zl
1981
MEASUREMENT
added
OF LYMPHOCYTE
to Instagel
determined
in scintillation
in a Packard
photon
emission
samples.
The
beta
was
minus
the
sucrose
as follows:
Lymphocyte
vials.
The beta radioactivity
in
volume
volume
895
spectrophotometer.
equivalent
lymphocyte
volume
VOLUME
The
the
supernatant
was calculated
of trapped
of
and
from
supernate
was
quenching
the
pellet
aspirated
mA
cytocrit
measured
by
at a flow
was set at 6 and
Coulter
electric
(cu sm/cell)
Lymphocytocrit
=
volume
Cell
(jl)
count
Trapped
-
volume
(.tl)
increase
pulse
height,
volume
through
enters
must
the
the
is
related
cell
1 .66 mmole
suspension.
suspension
One
was added
on an analytical
and
of 6 x I0
containing
ml of the cell
The
cells
50
beta
z1 TC-199
and
were
reweighed
The
the
dispersed
cell
insides
the
KOH
added.
This
incubated
sample
of
beta
in the
samples
were
prepared
by adding
the cell
pellet
the
pellet
surface
performed
and
to measure
evaporated
during
resulted
This
by medium
on
radioactivity
from
the
that
the
total
19%
of the
lated
from
did
and
pellet
wet
weight
the
since
Particle
volumes
particle
volume
were
particles
passing
ZBI
evaporation
weight.
was subtracted
for
‘
about
was calcu-
weight
of I .06’
The
supernate
-
by the formula:
count
(no/pellet)
Volume
and
cum/ml
integrated
from
this
particles
most
base
+
reproducible
to the mean
was equipped
under
integration:
with
or modal
with
particle
oscilloscope.
of the
current,
the instrument
electrolyte
(suspending)
A
parameters
Kfe
=
where
K is a constant.
function
of the
of the
resistivities
and the particle
P2’ A, and
I,
the
blood
shape
f,
value
off,
that
volume
this signal
or
to
used
reflects
“electronic”
evaluate
K
determine
(Reference
12
of electronic
is
in
the
V.
of operation
shape
will
erythrocytes
the range
ofvalues
electronic
particle
have
with
=
flow.’2
will
A prolate
aperture
havef.
deform
to the
has
good
a
For
value of 1.5O.I23
J
a smaller
ellipsoid
its longer
calculated
shapes.
with
axis
value,
as it
a large
parallel
axial
to the
that
normal
as they flow into analyzer
apertures
prolate
axis orienta-
An
encountered
been
some
1.0. It has been observed
ellipsoids
with
experimental
of I .04 for erythrocytes.
volume
for
and experimental
particle
are approximately
in a value
particles
experimentally
ellipsoid.
in the
human
parallel
various
spherical
of flow,
resulted
for
a prolate
oriented
tion
the principals
confirmed
direction
they
and
of
are in turn
is the signal
v
that
correctly
be determined.’2
has a theoretical
A deformable
that
to
particle,
a function
factors
“effective”
order
instrument)
describes
f,
rigid spheres,.!,
ratio,
must
and
into
the
only
is
Since
the
the usual operating
these
show
the
In
V,.
to the
a
i5
aperture,
analyzers.)
of
theory
deforms
f,
equations
is called
particle,
review
The value
from
which
V.
Under
in the orifice;
f,
factor,
in the
medium
ratio.
suspensions,
these
2 (calibrate
recent
orientation
deformability.
volume,
the
equation
correct
suspending
cell
(2)
conductivity
of the
and orientation
cell
of
and
and
on the particle
product
volume
shape
V
shape
to orifice diameters
with
particle
The
particle
(1-1.5)
long
determination
Thesef.
when
of
values
calibrating
so
f,’6
illustrate
and
using
analyzers.
Calibration
studies
electronic
counter
and
by Electronic
a Coulter
Channelizer
the
(1)
V
Calibration
of the Instrument
for Measurement
Latex
Spherules
and Red Cell Volume
2
x 10
(g/ml)
from
by the
RESULTS
determined
analyzer
derived
was
n/2
and
compared
were
Channelizer
calculated
best
that
volume
(total
on
were
accounted
lymphocyte
but
medium
this
weight
and
ofLymphocyte
volumes
the
and
Counting
Particle
on
to
studies
water
.
Measurement
buffer
pellet
Ife
resistivity
=
(g)/cell
density
voltage
V,
and
particle
Blank
to assess the radioac-
represent
cell density
for
was removed
Further
water
and
of photon
containing
walls.
not
The
(cu Mm/cell
Cell
volume,
particle
ing medium
to measure
was
mm
samples.
within
of lymphocytes
lymphocyte
volume
quenching
pellet
radioactivity
radioactivity.
the
aperture
The
area, andf.
dependent
45 mm.
to Instagel
The
process,
did
pellet
pellet
of the suprapellet
not represent
‘4C-sucrose
total
Lymphocyte
Cell
that
filter
wet
30
specific
I is the electric
conditions
of
0.4 ml of 2.5 N
added
served
tube
weighing
the wet weight
wet weight)
the
cell
and
trapped
the proportion
in radioactivity
the
current.
is the aperture cross-sectional
is a shape and conductivity
factor. For a given suspend-
ratio
10
with
20#{176}Cfor
-
supernate
blank
not
the
and
a ‘4C-sucrose
This
dried
at 37#{176}C
for
supernate
sedimentation.
as before.
at
was
radioactivity.
identical
treated
was weighed
with
15 mm
was
contributed
of this
measurement
The
frozen
500-.tl
was
tivity
per
at 900 g for
were
tubes
at 37#{176}C
for
emission
the pellet
and
A
after
that
for
balance.
water
digest
the
microliters
tube
tubes
The
dispersed.
measurement
across
the constant
j
of
was added
sedimented
fluid
of the
analytical
The cell lysate was thawed
thoroughly
conical
were
blotted.
in 1 .6 ml distilled
drop
a constant
nonconducting
AB
microcuries
sucrose
scintillation
surfaces
on
Three
20%
was added with 500 l of 0.5 N KOH
to Instagel
radioactivity.
paper
with
and eighty
is the
P2
medium,
in TC-l99
cells/mI.
to a 2.5-ml
balance.
From
unlabeled
hundred
mm and 50 sl of the supernate
and
Volume
Density
was prepared
at a concentration
‘4C-sucrose
a
equation:’2
where
plasma
to
with
When
voltage
4v
suspension
threshold
was set at 2
(no/pellet)
ofLymphocyte
Wet Weight
A lymphocyte
channel
current
operates
aperture.
to maintain
x l09cujzm/z1
Measurement
Lymphocyte
base
at I 00. The
analyzer
the
aperture,
in order
v,
The
threshold
at 1.
particle
current
particle
volume
of 2.5 mI/mm.
and the amplification
The
‘4C-
rate
the upper
The
the
channel
attachment.
size
distribution
number
(n)
curve,
median
and
channel
threshold.
perspective
of
.tm aperture
ZBI
Particle
the
=
median
channel
channel
encom-
This
The
provided
volume
Coulter
and particles
analyzer
spherules
sm-Particle
(2) Coulter
generated
of accumulated
particle
measurements.
a 100 by 150
Model
the
when
Model
were
with
the Coulter
Model
ZBI
“Channelizer”
particle
volume
were
performed
with
3 particles:
(source
with a true, mean volume
Information
4C Standard
leah,
Fla.),
and (3)
particle
was studied
(330
mosmole/liter),
of
Services,
(Coulter
Los Altos,
Electronics,
( I ) latex
of 1 I 3 cu
Calif.),
Hia-
normal
human
red cells.
Each
in 3 media:
( I ) Coulter’s
Isoton
(2) isotonic
phosphate-buffered
saline
(PBS)
(286 mosmole/liter),
PBS (250 mosmole/liter).
and
(3)
hypotonic
SEGAL.
896
Table
1 . Sizing
of Latex
Spherules
Suspending
Medium
330
Median
channe I (10)
cupm/channel
The
median
1/amp
number
channel
1 on
=
of measurements
is expressed
Suspending
23.7
±
0.1
24.3
±
0.2
Median
7.12
±
0.05
7.15
±
.05
6.98
±
.05
Electronic
SE) and cu Mm/channel
electronic
is shown
particle
in parenthesis.
are indicated
volume
for
analyzer.
The buffer
Volume
spherules.
their
and
( V)
Since
volume
when
they are rigid,
is represented
isl.S,
Vis
these
Table
spherules
were
I were obtained.
record
values
iv values
but
into
“channels,”
to v,
last
row
The
number
osmolarity
finger
sized
When
equation
n,
is
2 becomes:
(3)
the
cu
tm/channel
volume
electronic
is changed
as the suspending
swelling
of the 4C
in
has
Coulter’s
Isoton
(330
mosmole/
ratio
of 0.97
is
reported
volume
(with
unknownf
a true volume
of 83 cu
of 1.0. The change
in
(f,)
medium
red cells.
Tab le 3.
V,
1
of
±
2.8
87.2
±
2.8
93.5
on
1 .05
(effective)
the
measurements
(mean
electronic
is shown
0.4
±
2.8
1.13
volume
Coulter
±
±
particle
SE) are
volume
in parenthesis.
The
as mosmole/liter.
red cells.
The red cells from eight
donors
were added
directly
from a
The
Sizing
(8)
channel
with
tonicity,
the
the
and
media
and were
particle
volume
electronic
K2 values
red
cell
volume,
obtained
volume
with
in
the
the
three
suspending
media
was
calculated
using
the
ideal
osmotic
swelling
law.’4 The law states
that
red cell
volume
is a linear
function
of the reciprocal
of the
medium
cell volume
data
according
to this
tonicity
range
experimental
red
of Evans
and Fung’5
were
law and a linear
relationship
tonicity.
plotted
in the
of our
The
suspending
media
was
obtained.
Red cell volumes
were also calculated
from the ideal
osmotic
swelling
law using the same
plot slope but a
value of 87 cu sm for the isotonic
cell volume.”
These
calculated
volumes
are included
The experimental
f values
V,/ V, where
V, the true volume,
cu jsm at 286 mosmole/liter’4
Fung’s
data.’5
From these data,
shape
factor
for normal
human
0.99
what
in Table
3.
were
calculated
from
was taken either
as 87
or from
Evans
and
it is apparent
that the
red cells is between
and 1.1, and that its precise
we take as the true erythrocyte
off
equal
reasonable.
tonicity
median
(calculated
of Hum an Red Blood
to I .04
for normal
red
value
is sensitive
to
volume.
A value
cells
appears
to be
6
Cells
Suspending
Medium
Osmolarity
286
250
12.3
±
0.09
13.5
±
0.03
87.6
±
0.6
96.5
±
0.2
14.8
±
0.2
±
1.4
red cell
volumecu,um
True volumes,
Evans
cu zm,
based
and Fung data
factors,
Evans
1’,,
based
on true volumes
87cumat286
channel
and
electronic
studied
(effective)
is shown
89.3
97.4
79
87
105.4
95
0.98
0.99
0.98
1.11
1.11
1.09
.
from
and Fung data
of red cell populations
103.3
on
87cuimat286
median
80.5
stick to the three
suspending
individually
with the electronic
330
The
1
0.97
is expressed
suspending
and equation
3.
red cell standard
particle
factor
number
0.4
and electronic
=
human
healthy
medium
K2V,
sm is the electronic
Shape
±
latex
spherules)
are tabulated
in Table
3. Since
the
true volume
of a human
red cell varies with suspending
value)
or else the
m
and a shape
Electronic
1 2.2
v
sorts
the individual
channel
number,
is said to have a volume
of 83 cu
V, the true particle
volume,
or V,
channel
0.4
rather
the
4C Standard
is designed
for use with
solution
as the suspending
medium
liter).
In this medium,
the volume
essentially
unity,
indicating
that the
Median
±
analyzer.
I shows
volume
reflects
channel
1 /amp
250
3.4
1 1 .3
cu zm
for
Normal
different
the electronic
volume.
We used the calibration
factors
determined
with the latex spherules
to obtain
V values
in the three
different
suspending
media
(Table
2). A
volume
ratio can be calculated
as V/83
cu sm. The
of 83 cu
V,J83
Osmolarity
286
in
not
the data
The
Coulter
4C Standard
tm, which
may be
Medium
sized
electrically,
the data
Since
the instrument
does
from
Standard.
calculated
Coulter
4C
l70cuum.
cu zm
LICHTMAN
not
=
in Table
( I /K2)
do
placed
in media
of different
their effective
or electronic
by V
f V. In this case,f.
K2fV=
=
spherules
Vis
and
n
The
latex
Il3cuumandthus,
proportional
ratio
analyzer.
buffer
change
tonicity
volume
volume,
indicated
osmolarity
(6)
The median
The
as mosmole/liter.
Latex
channel
AND
of 4C Standard
330
0.1
±
Sizing
250
±
Coulter
2.
Osmolarity
286
23.8
(mean
the
Table
COKELET,
volume
(mean
in parenthesis.
±
SE) are indicated
The buffer
osmolarity
for
1 /amp
=
is expressed
1 on
the
Coulter
as mosmole/liter.
electronic
particle
volume
analyzer.
The
MEASUREMENT
OF
LYMPHOCYTE
897
VOLUME
Table
Exp.
No.
Measurement
of Lymphocyte
Trapped
Volume
(z1)
3.5
9.28
1.42
15.3
225
3.0
8.39
1.77
21.0
221
3
6.1
2.7
5
3.9
Mean
SE
t volume
effective
22.2
224
14.8
203
11.0
2.54
23.0
217
3.84
10.5
2.12
19.3
218
0.60
1.9
0.52
6.44
calculated
fro m the
equation
shown
in Materials
Volume
The
V,
volume
only
if the
of human
shape
blood
f,
factor,
lymphocytes
is
can
be determined
calibrated
with
only if the particle
volume
a particle
having
the same
analyzer
is
shape
factor
or if the
factors
particle
shape
the lymphocyte
cases
(such
as
are
latex
from
measured
independent
included
volume
by a lymphocytocrit
of the
volume
net lymphocyte
was determined
populations
lated
from
trapped
the
true
and
measurements
lymphocyte
methods
measurement
at
286
of
method
volume,
V,
mosmole/liter.
the
packed
and
cell
measurement
wet weight.
The lymphocyte
from the lymphocytocrit
in S
(Table
volume
experimental
the
by two
These
cell
calibrating
known.
Except
for a few special
spherules),
the shape
factor,
f,
must be determined
of Vand
V,.
have
for the
4). The
cell
lymphocytocrit
(see
volume
was
volume
Materials
and
calcu-
minus
the
Methods).
The
percentage
of trapped
volume
in these
studies
averaged
I 9 ± 0.4. The mean lymphocyte
volume
in these
studies
was 218 ± 4 cu tim. Alternatively,
lymphocyte
volume
was determined
from the cell pellet weight
and
lymphocyte
was calculated
wet
density
from
weight.
(Table
5). The
the total weight
The
percentage
cell pellet
weight
minus
the superof
Table
5.
supernate
Measurement
wet
of Lymphocyte
Total
Exp.
No.
and
0.4
4
Methods.
weight
volume
presented
above
indicate
that a particle’s
V. can be calculated
from its electronic
or
volume,
known.
3.91
0.95
was
17.6
ofLymphocyte
The data
volume,
was
17 ± 2.6,
by weight/density
and
the mean
lymphocyte
was 203 ± 3 cu .tm. The
mean
volume
from these
compared
to the electrical
mosmole/liter)
measured
two methods,
210 cu zm, is
volume,
289 ± S cu j.tm (286
in the same
lymphocyte
populations
using
latex
spherules
particle.
These
measurements
are
ferent
(p < 0.001).
Since
the shape
for lymphocytes
In practical
terms,
simplest
method
for
lymphocyte
volume;
analyzer
is calibrated
is the ratio 289/210
or 1.38.
the knowledge
off
permits
the
the electronic
measurement
of
that
is, the
particle
volume
with latex spherules
in isotonic
Ve/V,fc
buffer
volume,
(286
mosmole/liter)
Ve. The
Ve for
directly
volume,
can
in the same
V, is calculated
be calibrated
of Coulter
with
the
using
lymphocytes
buffer.
from
also
by
using
4C Standard
same
results.
lymphocytes
sized
true
The
tion
may
was
volume
lymphocyte
instrument
the
electronic
volume
or normal
human
red
The
median
channels
in Isoton
is taken
as a change
shape
factor
of I .38
their
electronic
is determined
The
V,/f.
(330
mosmole/liter)
(286 mosmole/liter)
and PBS (250
shown
in Table
6. When
the change
lymphocyte
as the standard
significantly
diffactor,fe,
equals
in lymphocyte
volume
is used
to determine
at each
osmolarity,
an
and the
the true
8% reduc-
Volume
Supernate
by Weight/Density
Wet
Weight
1mg)
Weight
mg)
Percent
Supernate
Lymphocyte
Volume
(cu Mm)
1
7.49
20.1
4.22
21
8.29
22.4
5.13
23
196
3
6.67
18.9
3.92
21
211
200
27.9
3.82
14
204
5
6.70
15.9
0.93
6
211
6
9.56
24.3
4.67
19
194
Mean
8.30
21.6
3.78
17
203
SE
The lymphocyte
1.7
0.71
volume
was calculated
from
the equation
0.60
shown
in Materials
are
channel
from 210 to 194 cu zm is observed
in Isoton.
This
not be an exact calculation,
since the shape
factor
determined
at
physiologic
osmolarity,
286
Cell Count
( x 1 0’ Cells)
11.1
cells
of
PBS
mosmole/liter)
in median
2
4
weight.
Lymphocyte
Volume
(cu Mm)
Percent
Trapped
1
Measurement
nate
by Lymphocytocrit
2
4
We
Volume
Lymphocytocrit
Volume
(z1)
Cell Count
lx iO’ Cells)
The lymphocytocri
true
4.
and Methods.
3
2.6
Cell pellet
wet weight
=
Total
weight
-
Supernate
wet
SEGAL. COKELET.
898
Table
Sizing
6.
of Human
Blood
Lymphocytes
Suspending
Medium
Osmolarity
-0
Median channel (9)
Electronic
37.4
266
volume
286
0.4
±
2.8
40.4
289
194
Truevolume
220
120
20C
100
C
0
±
LICHTMAN
a
330
)lsoton)
AND
250
±
0.4
±
2.9
44.0
307
210
±
0.5
±
3.5
80
18C
00
229
I-
The median
channel
indicated
for
1 /amp
analyzer.
The
number
parenthesis.
The
calculating
factor
1 on
=
of
buffer
was
1 .38
(effective)
the
Coulter
lymphocyte
mosmol/liter),
assumed
SE) are
±
particle
studied
is
the
value
lymphocyte
of the
be somewhat
altered
60
16C
45
volume
shown
shape
In
C)
shape
0
>
U
factor
0
at 330
#{176}
-e
in
as mosmole/liter.
that
This
may
(mean
electronic
is expressed
it was
at all osmolarities.
at 286
volume
populations
osmolarity
the true volume,
(measured
at 250
and electronic
and
mosmole/liter.
A1
.,‘J
14C
0
0
-0
12C
0.
20
E
-J
100
A
2#{212}0225
175
mosmole/liter,
andf
(330 mosmole/liter).
use of osmotically
may
be slightly
The
inactive
standardize
the
motically
responsive
altered
reduction
particles,
instrument
for
in Isoton
results
from
latex spherules,
measurement
Lymphocyte
the
to
of os-
lymphocytes.
250
275
300
Volume
(pm3)
Fig. 1 .
The lymphocyte sodium. potassium. and sodium plus
potassium concentrations
are plotted against lymphocyte volume.
The figure shows data calculated from a cell potassium of 25
fmole/cell
and a sodium
as 78% of the lymphocyte
of 3 fmole/cell.’7”
volume.’7
Cell water
was
taken
DISCUSSION
The accuracy
tronic
particle
factors.
These
ment, the area
characteristics
of volume
determinations
on an elecvolume
analyzer
is dependent
on several
include
electronic
features
of the instruand length
of the aperture,
and the flow
and deformability
of the particle
used
as a standard
and the
evaluated
the effects
particles
by calculating
particle
to be sized.
We have
of (I ) the deformability
of
the shape
factor,
f, and (2)
the buffer
osmolarity
on the
blood
lymphocyte
volume.
however,
to the measurement
The
precise
determination
particular
importance
in
measurement
of human
These
findings
apply,
of any cell’s volume.
of cell volume
is of
transport
and
metabolic
volumes
volume
22
from
1 75 to 300 cu tm.
At a lymphocyte
of 1 75 cu .tm, the Na and K concentrations
are
and
183
mmole/liter
cell
water,
respectively.
contrast,
at a volume
of 300 cu m,
the
concentrations
are 12 and 108 mmole/liter
Thus,
from
differences
measurement
In
Na and K
cell water.
in the apparent
cell volume
resulting
errors
can account
for a difference
in cell concentration
of a constituent
The reported
values
for
from
180 to 280 cu tm.’9
of up to 80%.
lymphocyte
volume
range
Such
variation
would
be
predicted
without
if the volume
measurements
were
regard
for the deformability
characteristics
the cell
to be measured
and
the standard
particle
made
of
used
experiments
in which
cell volume
contributes
to the
estimation
of the
distribution
space
of internal
contents.
Cell volume
is a rapid,
easy, accurate,
and
reproducible
basis for calculating
the cell water
when
compared
to more
tedious
methods.
Cell
water
or
volume
can serve as the denominator
for calculation
of
to calibrate
the instrument.
For example,
if the shape
factor
is ignored
and the instrument
is calibrated
with
latex spherules,
the lymphocyte
electronic
volume
in
isoosmotic
medium
is 193 cu sm. In contrast,
the
lymphocyte
electronic
volume
is 276 cu sm if the
instrument
is calibrated
with
the Coulter
4C Stan-
the concentration
or content
of internal
influence
of cell volume
on the apparent
dard.
This
range
of volumes
corresponds
to the
reported
values
for lymphocyte
volumes;
moreover,
the
lowest
values
were
measured
when
latex
spherules
were used to calibrate
the electronic
particle
sizer3’4’6
of internal
monovalent
solutes
cation
concentration
180
mmole/liter
Although
measuring
volume
shown
content
has
is exemplified
concentration.
been
cell
substance.
The
concentration
by its effect
on
The cell potassium
reported
between
water
in mammalian
1 10 and
cells.
some variation
may be due to difficulties
in
the cations
themselves,
the influence
of cell
on cation
concentration
in Fig. 1. In this analysis,
of 25 fmole/cell
cell’7”8
have
monovalent
been
cation
and
Na
used
to calculate
concentration
may
the
be marked,
lymphocyte
content
the
at
of 3 fmole/
lymphocyte
lymphocyte
as
K
and
the
highest
for calibration.8’9
ignored
and true
Two
shape
group
groups
values
when
red
blood
cells
In these
studies,
shape
volume
was not measured.
of
investigators
have
were
used
factor
was
considered
factor
in the sizing of human
lymphocytes.
concluded
that
the lymphocyte
behaved
the
One
as a
rigid sphere
on the basis ofdirect
microscopic
observations of cells flowing
through
the sizing
aperture
and
concluded
that lymphocyte
volume
was 180 cu
MEASUREMENT
They
OF
did
not
lymphocyte
The other
LYMPHOCYTE
provide
899
an independent
measurement
f
volume,
however,
to verify
an
group
used data
calculated
from
ments
of cell diameter
determination.2
They
shape
factor
to be that
calculated
a lymphocyte
By
VOLUME
using
two
lymphocyte
as the independent
volume
considered
the
lymphocyte
of a rigid sphere
(J
I .5) and
volume
of 195 cu tm.
=
independent
volume,
of
of I .5.
measure-
methods
we have
value of 1.38. This
slightly
greater
than
arrived
to
measure
at a shape
factor
An additional
but
problem
is introduced
molarity
of Isoton
results
in about
an 8% decrease
of
lymphocyte
volume
measured
in isotonic
medium
with
latex spherules
as the standard
particle.
results in a lymphocyte
volume
that reported
by Ben-Sasson
and
coworkers.2
The difference
between
a shape
factor
of
I .38 and I .5 is small,
and the important
insight
is that
lymphocyte
volume
is about
200 cu m rather
than the
higher
values
of 250-290
cu sm.
less critical
in the measurement
of cell volume
if the commercial
diluting
solution,
Isoton,
is used
to suspend
the
particles
for measurement
and an osmotically
inactive
particle
is used as a standard.
Isoton,
despite
its name,
has an osmolarity
of 330 mosmole/liter.
The hyperos-
ACKNOWLEDGMENT
We
thank
Dr.
Regional
Red
pheresis
residues.
and
Valerie
these
Jacob
Nusbacher
Cross
Blood
Elizabeth
Black,
and
Center
Dr.
for
Kearney
a student
Joanna
Heal,
supplying
provided
at Keuka
Rochester
human
technical
College,
also
plateletassistance
participated
in
studies.
REFERENCES
I
.
Thom
sizing,
G, Lewis
York,
Academic,
Ben-Sasson
sizing
and results by improved
in Izak
gy. New
2.
R: Method
(eds):
1972,
5, Patinkin
of particles
84:205,
SM
Modern
electronic
blood-cell
Concepts
in Hematolo-
Doljanski
F: Electrical
rapid
p 191
NB,
IV.
Lymphocytes.
J Cell
from
Physiol
1974
P, Chanana
of human
lymphocytes
of nuclear
volume
and
HB,
Nielsen
AD,
Sipe CR,
in culture:
cell
Crokite
EP: Proliferation
Determination
number.
Nouv
I 2.
by measurement
Rev
Fr Hematol
in
20:545,
volume
spectroscopy.
5.
HG:
membrane:
Applying
to measure
rapid
Braylan
Herman Ci:
neoplastic
Westring
of human
8. Segel
Nusbacher
of human
9.
Studies
Sipe
J Immunol
The
changes
10:135,
volume.
cells.
Cytol
DW,
Ladinsky
Proc
Lichtman
JL,
Feick
Soc Exp
MA,
J: Plateletpheresis
41:201,
Med
BR,
A source
1969
MacPherson
JL,
quantities
blood lymphocytes.
Transfusion
16:455, 1976
CR,
Chanana AD, Cronkite
EP, Gulliani
GL, Joel DD:
on lymphocytes,
XIII.
Nuclear
volume
measurement
J Haematol
PF,
New York,
Naaman
EA,
Waterman
CS,
Improved
Mendelsohn
Wiley,
granulocytes
97):77,
1968
normal
Blood
sizing
human
34:591,
1969
(Coulter
ML
sizing),
(eds):
Flow
1979, p61
5, Doljanski
in suspensions.
II.
F, Nadav
Experiments
E:
with
1969
and
Related
Phenomena
(rev
ed).
New
I 971
Fung
geometry.
pulse
i, Ben-Sasson
J 9:1415,
& Stratton,
of
by size.
resistance
E: Hemolysis
Evans
SL:
separation
and
21 (Suppl
Improved
measurements
Microvasc
YC:
Res 4:335,
1972
Atkinson
EE
measurement
Jr.
Wilkins
B Jr.
of erythrocyte
of
Fischer
volume
the
CL,
distribu-
tion by aperture-counter
signal analysis. Clin Chem 21 :1 201, 1975
17. Segel GB, Lichtman
MA: Potassium
transport
in human
distribution
I 3 1 : I 077,
of large
16.
Kimzey
The
of particles
Biophys
Ponder
erythrocyte
B:
Mullaney
NB,
sizing
Grune
I 5.
1977
1978
P: The volume
Biol
Gordon
residues:
21:96,
5K, Berard CW,
of normal and
Invest
and classification
MR.
spheres.
York,
Lab
Cassen
and Sorting.
Grover
14.
computer
Scand
cells
J Clin
V: Electrical
Melamed
Electrical
lymphocyte
and a hybrid
Acta
Cancer
by
fraction.
of mononuclear
Scand
by density
Cytometry
rigid
the
BJ, Jaffe ES, Sanders
and DNA distributions
lymphoid
studied
1979
of
size analyzer
in cell
lymphocytes.
GB,
blastogenesis
permeability
a particle
RC, Fowlkes
Cell volumes
human
Lymphocyte
Scand
Hempling
6.
V:
proliferative
Isolation
RM,
Kachel
13.
4. Steen
A:
blood.
I 1. Zucker
I978
7.
Boyum
human
leukocytes
3. Chandra
to estimate
1976
10.
D, Grover
in suspensions.
approach
16:196,
as a
blood
lymphocytes
58:1358,
18.
and
blood
treated
with
phytohemagglutinin.
J Clin
Invest
1976
Segel
GB,
potassium
lymphocytes.
Simon
transport
J Clin
W,
in
Lichtman
MA:
Regulation
phytohemagglutinin-stimulated
Invest
64:834,
1979
of sodium
human