From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
Ficoll
Flotation
for
the
Separation
Leukocyte
By
P.
T SHOULD
BE
differences
high
B.
POSSIBLE
in their
density
fluids
from
peripheral
in this manner,
labor
involved
the viability
has
describes
further
the
CurrS
cells
at least
acacia3
has
and
been
the media,
isolated.6
We
for
and
AND
K.
K.
Cnnou.
of different
Centrifugation
successful,
in part,
bovine
criticized
types
by
of whole
in separating
albumin1’2’5
because
utilizing
blood
against
leucocytes
have been
used
of the cost and
and doubts
have
been
expressed
have
reported
previously7
the
as to
use of
the isolation
of leukocytes
from
peripheral
appears
to be nontoxic
to cells. The present
experiences
illustrate
Huiny
gravities.
Gum
method
in preparing
the cells
Ficoll
as a density
fluid
It is inexpensive,
stable
which
been
Blood
Types
to separate
specific
blood.15
but the
of
J.
NoBu,
of
with,
usefulness
and
of Ficoll
some
far
modifications
separation
of cells
blood.
paper
of
the
method,
by
the
flotation
method.
METHODS
Ficoll,
present,
an inert,
high
and lyophilized.
#{176}
solution:
this
was
reduced
AND
molecular
weight
For dialysis,
the
to
OBSERVATIONS
polysucrose
Ficoll
was
approximately
20
per
was
made
cent
dialysed
to remove
the NaCl
up as a 50 per cent aqueous
during
dialysis.
Concentrations
much
greater
than this did not lyophilize
well, and in place
of a light fluffy material,
the
Ficoll was recovered
as a solid “glassy”
substance
which
appeared
to contain
water.
The
recovered
Ficoll was stored
in tightly
stoppered
glass bottles
to prevent
absorption
of water.
A 10 per cent solution
of Ficoll in Seligmann’s
balanced
salt solution
(SBSS),fs
has a specific
gravity
of 1.039.
Provided
the specific
gravity
of the lyophilized
material
was checked,
no
detectable
variations
have been noted in the different
batches
of Ficoll.
Appropriate
concentrations
of Ficoll
were made
up as needed
by dissolving
the Ficoll
in
SBSS. The solution
was filtered
through
a Millipore
filter of 0.45 t pore size, and distributed
#{176}Ficoll obtained
Qu#{233}bec,Canada.
fSBSS-7.65
from
gm
NaCl,
Pharmacia
0.20
gm
(Canada)
KC1,
1.50
Ltd.,
gm
100
Place
NaCO9CH:,
Cremazie,
0.05
KH2PO4,
0.70 gm NaHCO1,
1.0 gm glucose,
0.003
gm ascorbic
1000 ml. pH of the solution
7.3.
Cellulose
nitrate
tubes
1 in. diam. X 3 in. Spinco
Division,
Palo Alto, California.
gITI
acid,
Montreal
11,
NaH2PO4,
0.10 gni
twice
distilled
water
Beckman
Instruments
Inc.,
From
the Collip
Medical
Research
Laboratory
and Department
of Anatomy,
Health
Sciences
Center,
University
of Western
Ontario,
London,
Ontario,
Canada.
This work was supported
by the National
Cancer
Institute
of Canada.
First submitted
May 3, 1967; accepted
for publication
July 6, 1967.
P. B. NOBLE,
B.Sc:
Research
Fellow,
Collip
Medical
Research
Laboratory,
University
of
Western
Ontario,
London,
Ontario,
Canada.
J. H. Cui-rs,
PH.D.
: Associate
Professor,
Department
of Anatomy
and Cancer
Research
Laboratory,
University
of Western
Ontario,
London,
Ontario,
Canada.
K. K. CARROLL,
PH.D.:
Professor,
Department
of Medical
Research,
University
of Western
Ontario,
London,
Ontario,
Canada.
66
BLOOD,
VOL.
31,
No.
1
(JANUARY),
1968
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FICOLL
in
FLOTATION
FOR
SEPARATION
OF
LEUKOCYTE
67
TYPES
nitrate
tubesl
of one
inch
diameter.
In all cases,
9 ml. of Ficoll was used to
recovery
of the cell layer. Quantities
less than this resulted
in the cell layers
being
too near the bottom
of the tube to permit
convenient
recovery.
cellulose
facthtate
formed
The methods
described
below are those employed
for
blood, but the method
is applicable
to other species.
We
from human
and from rat peripheral
blood.
Modifications
made and are described
below and outlined
in Figs. 1 and
Stage
I: Separation
of Leukocytes
from
isolating
at
10800
an
g for
30
mm.
The
entire
from
chicken
leucocytes
have
been
Erythrocytes
Initially,
heparin
was used as the anticoagulant,
and possibly
loose clumping
of the leukocytes.9”#{176} To overcome
clumping,
5
directly
into 5 ml. of Seligmann’s
solution
containing
5 mg. of
The blood was gently
mixed
and layered
over 9 ml. of 35 per
carried
same
As
face.
cells
leukocytes
have used it to separate
of the original
method
2.
procedure,
including
as a result
of this, there was
ml. of blood
were collected
EDTA
as the anticoagulant.
cent Ficoll,
and centrifuged
withdrawal
of the
blood
sample.
was
out at 4 C. All reagents
and equipment
used had been
chilled
previously
to the
temperature.
can be seen in Fig. 1, a grayish-white
band formed
just below
the Ficoll:
plasma
interclearly
separated
from the underlying
erythrocytes.
This layer of discretely
dispersed
was recovered
by puncturing
the side of the tube just below
the buffy
layer,
using
18
gauge
needle
and
syringe.
The
cells
were
washed,
resuspended
in
Seligmann’s
solu-
tion and examined
by phase
contrast.
The cells of this layer,
shown
in Figs. 2 and 3 of
Plate
1, consisted
of lymphocytes,
monocytes
and thrombocytes
with
only an occasional
erythrocyte.
The granulocytes
remained
intimately
mixed
with the red cells of the upper
portion
of the erythrocyte
layer.
and it was not possible
to separate
these
two cell types
further
by Ficoll flotation.
The above
method
successfully
separated
lymphocytes
and monocytes
in a high degree
of purity.
Because
of our failure
to recover
granulocytes,
clumping
of all leukocyte
types
was promoted
by withdrawing
5 ml. of blood
into 1 ml. of Hank’s
balanced
salt solution
(HBSS)11
which
contained
100 units of heparin.
The blood
was layered
over 9 ml. of 35
per cent Ficoll in SBSS and centrifuged
at 1000 g for 30 minutes
at 4 C.
The leukocytes
separated
as a cohesive
sheet
just below
the Ficoll:
plasma
interface
as
shown
in the upper
portion
of Fig. 2. The cells could be removed
by gentle
suction,
using
a syringe
and a large bore needle
having
a long bevel.
This layer contained
all the leukocyte types mixed with thrombocytes
and a few erythrocytes.
Stage
II:
The
further
Separation
lower
of Individual
portion
separation
of Fig.
of
the
Leukocyte
2 shows
leukocyte
the
types.
Types
various
The
cells
Ficoll
were
preparations
washed
used
and
to bring
redispersed
about
in
SBSS
and
layered
over a single-step
preparation
consisting
of 9 mIs. of 28 per cent
Ficoll
in
SBSS (Tube
1, Fig. 2). The tubes were centrifuged
at 300 g for 15-20
mm. at 4 C. Lymphocytes,
monocytes
and thrombocytes
formed
a layer
at the Ficoll
interface
(Tube
1, a).
Granulocytes
and the few contaminating
erythrocytes
and thrombocytes
clumped
at and
just
below the interface
and could not be separated.
Although
some
of the granulocytes,
contaminated
with a few red cells and thrombocytes,
were
present
in the lower
portions
of the tube, additional
centrifugation
served
only to throw
all cell types
to the bottom
of
the tube.
In order
to avoid the abrupt
interface
between
the Ficoll
and the overlying
cell suspen-
sion, further
modifications
were tried. Three ml. of Seligmann’s
solution
were layered
over
9 ml. of 28 per cent Ficoll and allowed to equilibrate
for 4 hr. or for 24 hr. at 4 C. These
are
shown
as tubes
2 and
prepared
also, by layering
Ficoll in successive
layers,
2). The
Good
achieved
cells
were
layered
3 respectively.
in Fig. 2. Nonlinear
continuous
gradients
were
5 cc portions
each of 30 per cent, 25 per cent and 20 per cent
and allowing
them to equilibrate
for 48 hrs. at 4 C. (Tube
4, Fig.
over the gradients
and centrifuged
at 300 g for 15 mm. at 4 C.
separation
of lymphocytes
by any of the latter three
and monocytes
from granulocytes
and erythrocytes
was
preparations.
The cells so separated
are shown
in Figs.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
68
NOBLE
ET
AL.
Blood
taken
into SBSS
contoining
5mg EDTA.
Layered
on 9 mIs Ficoll, centrifuged
at 1000 g
for 30 min.at
4#{176}C
HETEROPHILS
Fig.
and
RBC
1.-Leukocyte
LYMPHOCYTES,
MONOCYTES
and THROMBOCYTES
separation
using
Seigmann’s
balanced
salt
solution.
10 and 11, Plate 2. Discontinuous
lighter
erythrocytes,
not previously
discretely
dispersed
throughout
settled with the heavier erythrocytes
Ficoll
gradients
separated
an additional
distinct
band
of
seen. This band
contained
a number
of granulocytes
the
red
cells.
The
remainder
of the
granulocytes
to the bottom of the tube, and were present
in coarse,
loose clumps.
The granulocytes
could be freed from
the few contaminating
red cells by
“paradoxical
sedimentation,12”3
as shown in Fig. 12, Plate 2.
Although
evaluation
of the morphological
appearances
and the results
of viability
studies
are not yet completed,
it can be stated
that
the leukocytes
separated
by flotation
over
Ficoll retain morphologic
integrity,
both on stained
smears
and in living
cells examined
by
phase microscopy.
Staining
with Trypan
blue has shown an excess of 80 per cent of the
cells to exclude
the dye. When
warmed
to 37 C on a warm
stage,
the cells showed
good
ameboid
activity,
a phagocytic
and
phagocytosis
monocyte
is seen
of
in Plate
thrombocyte
1, Fig.
particles
by
monocytes
were
seen.
Such
6.
DISCUSSION
Ficoll
appears
to offer a medium
ically
intact,
viable
leukocytes
from
studies,
it was
thought
that
separation
be achieved
only by preventing
appears
to be so excessive
with
was
surmounted
balanced
salt
by collecting
solution
and,
suitable
whole
for
blood
of the
the
by
separation
cell flotation.
individual
leukocyte
the nonspecific
clumping
flotation
methods.’3”4
The
the
avoiding
initial
blood
heparin.
sample
Leukocyte
of morphologIn our early
type
could
of leukocytes
which
problem
of clumping
directly
into
separation
Seligmann’s
was
then
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
FICOLL
Blood
FLOTATION
FOR
SEPARATION
taken
into HBSS
9 mis. Ficoll,
centrifuged
OF
LEUKOCYTE
containing
100 units
at 1000
Lymphocytes,
heterophils,
layered
on 9 mIs. Fico!!,
69
TYPES
of heparin.
g for
30
Layered
mm. at 4#{176}
C.
monocyte s, thrombocytes.
Dispersed
in SBSS
centrifuged
at 300 g for 15mm. at 4#{176}
C.
C
2
1
a-lymphocytes
and
b-light
erythrocytes
c-heavy
erythrocytes
Fig.
achieved
be
by
have
the
2.-Leukocyte
separation
flotation,
Since
same
3
4
monocytes
and
heterophils
and heterophils
Ficoll
recovered.
some
specific
but
of
using
only
the
gravities,
the
Heparin.
lymphocyte-monocyte
erythrocytes
no
BSS and
Hank’s
distinct
and
fraction
granulocytes
separation
of the
could
appeared
to
granulocytes
was
obtained.
When
gates
clumping
leukocyte
No
thrombocytes.
but nearly
cells
were
distinct
types,
separation
all of the applied
easily
dispersed
separation,
flotation
of leukocytes
of mixed
was
on
heparin
was
used
to separate
occurred,
the
in
were
which
of the
cells could
be
in Seligmann’s
used
to
the
encourage
various
white
cells
various
a few
leukocyte
recovered
solution.
types.
and
as aggrered
entities
as a single
In further
clumping,
leukocyte
separated
trapped
band.
trials
additional
cells
and
occurred,
These
of cell
Ficoll
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
70
NOBLE
Plate
1.-Peripheral
and thrombocytes.
rod-shaped
granules
kocytes
isolated
blood
(1
from
)
Phase
which
stage
from
the
contrast
made
I Ficoll
the
x
chicken
125.
showing
Chicken
cell
difficult
flotation
using
lymphocytes
heterophils
to photograph.
Seligmann’s
ET
AL.
heterophils
contain
(2
solution.
&
refractile
3) LeuNote
the
absence
of clumping
and the loss of granulocytes
and erythrocytes.
Phase
contrast
x 125. (4 ) A clump of leucocytes
isolated
from stage I Ficoll flotation
using SBSS
and heparin.
Phase
contrast
x 1125. (5) The same cells as in (4), showing
the
clumped
nature
of the cells.
Note
that granulocytes
( heterophils
) are present
in
large
numbers,
along
with lymphocytes,
monocvtes
and a few ervthrocytes.
Phase
contrast
X 125.
(6-8)
Morphological
features
of leukocvtes
isolated
by stage
I
Ficoll flotation.
(6) Monocytes.
One cell is shown
engulfing
a thrombocyte
(arrow).
Phase
contrast
x 1125.
(7) Large
monocvte
from stage I Ficoll using HBSS.
Phase
contrast
x 1125.
(8)
Heterophils.
These
cells show
typical
refractile
rod-shaped
granules.
Phase
contrast
x 1125.
(9)
Human
granulocytes
isolated
by stage
I
Ficoll flotation
using HBSS. Phase contrast
x 1125.
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FICOLL
FLOTATION
FOR
SEPARATION
OF
LEUKOCYTE
71
TYPES
Plate 2.-(1O)
Cells isolated
at stage II, band a. L = lymphocyte,
M = monoThe = thrombocyte
X 125.
Insert
shows
a monocyte
X 1125.
(11) Cells isolated
at stage II, band c X 125 H = heterophiles,
R = ervthrocytes,
Th = thrombocytes.
cyte
( 12)
Cells
from
stage
IL, band
erythrocytes
and reduction
bocytes
Insert shows two
In order
to show
prior to photography.
L = lymphocyte,
H
On
the
initial
below
the
Ficoll:
c after
heterophils
modifying
sedimentation.
H 125
H
=
Note
heterophiles,
cell detail
more clearly,
the preparations
were
The legend
is the same for all illustrations.
heterophil,
Th = thrombocyte.
35 per
serum
cent
the
Ficoll,
interface
original
the
Th
=
lack
of
throm-
H 1125.
the
but
on a lower
concentration
of Ficoll.
passed
through
the interface,
small
thus
paradoxical
of thrombocytes.
interface.
cells
separated
subsequent
It would
amounts
The
as a band
separation
appear
of serum
Ficoll
crushed
M
that
were
became
could
as the
carried
diluted
=
slightly
monocyte,
a few
be
mm.
achieved
erytbrocytes
along
also,
in
a narrow
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
72
NOBLE
zone
extending
ous
below
gradient
At
the
second
of
the
at
Ficoll:
medium
from
Ficoll
sufficient
separated
form
interface,
had
the
clumps
flotation,
of mixed
leukocytes,
to
a band
of
solutions
to plasma
by malaria,
found
sented
evidence
cytes.16
Vallee,
erythrocytes
men.
We
have
these
noted
contain
a band
that
and
blood
light
a high
Ficoll
type
granulocytes
from
Throughout
ferent
problem.
cell
small
the
these
clumps,
tamination
the
or
was
High
density
problem
that
ent
stages
of cell
by
small
constantly
numbers
altered
it did
as nucleated
cific
gravities
elements
of chicken
be
making
very
blood
close,
with
much
lighter
The absence
of
a short
linear
at
tended
interfaces
cells.
were
could
Ferrebee,’5
down
young
from
and
however,
in chicken
using
shed
their
In the
platelets,
lighter
albu-
on
human
blood,
studies
I.
of the
( platelets
gravity
)
as they
dif-
was
Thrombocyte
human
latter,
blood,
thrombocytes
did
a
formed
connot
pose
are
pres-
lymphocytes
in size. The speand thrombocytes
appear
to
of these
cell
does
present
not
of
contamination
and
approximating
lymphocytes
separation
of erythro-
Preliminary
at stage
cytoplasm.
in rat
and
parapre-
and
that
reticulocytes
do
from
the more
maour inability
to sepa-
of thrombocytes
their
specific
blood.
closely
monocytes,
rat
suggest
separated
explains
separation,
and
forms
cells.
by SBSS
the
to collect
noted
the separation
anemia,
after
flotation
separating
obtained
to
with
SBSS,
or by
separated,
in addi-
red
of polychromatic
erythrocytes
great,
failed
remained
they
cells
have
with
erythrocytes
proportion
broke
not
of
interface
lighter
lighter
Gibso&7
of patients
various
fractions
Thrombocytes
flotation
interface,
on the separation
of cells
from
bone
marrow,
form
a layer
of less dense
erythrocytes,
clearly
ture cells.
The presence
of lighter
erythrocytes
rate
Ficoll
to equilibrate
of preparation
of
continu-
liquids.
to obtain
differential
flotation
of erythrocytes
the parasites
in a layer
of lighter
red cells,
to show
Hughes
from
the
this
AL.
of leukocyte
types.
LymphoFicoll
interface,
immediately
density
met
the
This
a short
two
cells and granulocytes.
the establishment
of leukocytes.
allowing
gradients.
in fact,
of the
single-step
a high
cells
types
tion
dense
sitized
that
As the
by
become
junction
to permit
recovery
as a band
at the
so
junction.
be avoided,
either
making
discontinuous
and
and
mm.
and in contact
with a layer
of red
numbers
of erythrocytes
prevented
gradient
and
interface,
a few
stage
achieve
separation
cytes
and monocytes
above
large
the
extending
ET
types
more
the
same
difficult.
Mammalian
problem.
SUMMARY
A method
tation
over
for the separation
Ficoll,
is described.
ation,
and the leukocytes
integrity,
are motile
and
of leukocytes
The method
isolated
capable
SUMMARIO
Es
describite
un
methodo
pro
le
from
peripheral
blood,
using
fib.
is amenable
to considerable
van-
show
good
of phagocytosis.
preservation
of their
morphologic
sanguine
peripheric
IN INTERLINGUA
separation
le utilisation
de flottation
supra
Ficoll.
Le
adaptabilitate.
Le leucocytos
isolate
manifesta
phologic.
Ilbos es motil e capace
de phagocytosis.
de
leucocytos
methodo
un bon
ab
se distingue
preservation
con
per un alte grado
de
de br integritate
mor-
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
FICOLL
FOR
FLOTATION
OF
SEPARATION
LEUKOCYTE
73
TYPES
ACKNOWLEDGMENTS
Thanks
Barry
are
extended
Forbes
to
Miss
for photography
Tia
and
Brinkman
for
excellent
technical
assistance
and
to Mr.
l’utiiser
dans
art work.
REFERENCES
1. Fawcett,
D. W.,
and
Vallee,
B. L.:
Studies
on the separation
of cell tpyes
in sero-sanguinous
fluids,
blood
and
vaginal
fluids
by flotation
on bovine
plasma
J. Lab.
albumin.
Clin.
Blood
3:1055,
10.
Med.
39:354, 1952.
2. Vallee,
B. L., Hughes,
W. L., and Gibson
J. C.: A method
for the separation
of
leukocytes
from
whole
blood
on serum albumin.
Blood 1:82, 1947.
3. Spear.
F. : Separation
of leukocytes
from
whole
blood
by flotation
on gum acacia.
et
N.Y.
11.
12.
4.
tionation
of
by their
of cells
fowl
blood
buoyant
active
in
actions.
Ann.
N.Y.
Acad.
Sci.
elements
means
Med.
7. Noble,
tion
of
of
fraction
I.
of
blood
by
Vet.
Jour.
M., and
J. M#{233}thodesde preparation
leucocytaires
et de serums
cytaires
pour
Ann.
9. Fleck,
Separa-
15.
d’etre
Proc.
Soc.
p.
York,
1955.
Exp.
Biol.
Med.
9
Grune
&
Stratton,
1960.
J. W.,
Ferrebee,
Studies
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Blood
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1968 31: 66-73
Ficoll Flotation for the Separation of Blood Leukocyte Types
P. B. NOBLE, J. HARRY CUTTS and K. K. CARROLL
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