[CANCERRESEARCH37, 3876-3880,November1977]
Separation of Leukemic Cells into Proliferative and Quiescent
Subpopulations by Centrifugal Elutriation1
Harvey 0. Preisler,
Irene Walczak,
Joyce
Renick,
and Youcef
M. Rustum
Departmentof MedicineA (H. 0. P., I. W.,J. R., Y. M. R.Jand GraceCancerDrug Center(Y. M. R.J,RoswellParkMemorialInstitute, Buffalo, N@wYork
14263
SUMMARY
MATERIALS AND METHODS
Centrifugal elutriation was used to separate human acute
leukemia cells into proliferative and quiescent subpopula
tions. Ten bone marrow specimens and 5 peripheral blood
specimens were subjected to centrifugal elutriations. From
each patient, leukemic cell subpopulations were obtained
for which the [3H]thymidine labeling index differed by 10to 30-fold. In 6 of the marrow specimens and in 2 of the
peripheral blood specimens, cell subpopulations were ob
tamed for which the labeling index exceeded 20%. In 5
marrow specimens, subpopulations were obtained for
which the labeling index exceeded 40%. Preliminary studies
of the uptake of 1-f3-D-arabinofuranosylcytosine and 5-aza
cytidine failed to show any correlation between drug uptake
and the proliferative characteristics of the leukemic sub
populations.
Specimens for Study
Informed consent was obtained from each patient. Five
to 10 ml of bone marrow were aspirated from a posterior
iliac crest into a syringe containing 2 ml of 4% sodium
citrate as anticoagulant. Ten to 20 ml of peripheral blood
from patients with a WBC >30,000 were anticoagulated
similarly. The syringes were immediately placed into an ice
bucket, and the specimens were transferred to a sterile
plastic tube and centrifuged at 600 x g for 10 mm in an
International refrigerated centrifuge. The buffy coat was
then removed, and the residual erythrocytes were lysed by
suspension for 10 mm in a solution of 0.75% ammonium
chloride in distilled water containing a 2.06-g/liter Trizma
base (pH 7.2). Cells were recovered by centrifugation at
800 rpm for 8 mm, and the supernatant
INTRODUCTION
The neoplastic cells of patients with acute leukemia can
be subdivided on the basis of size into proliferative and
quiescent subpopulations (3, 4). The LI's2 of the former are
in the range of 40 to 50%, whereas those of the latter are in
the range of 0 to 1%.
At least some of the quiescent subpopulations can re
sume active proliferation (2, 8). These quiescent cells ap
pear to be relatively resistant to chemotherapy and hence
make remission induction therapy more difficult and may
be responsible for leukemic relapse (1).
Attempts have been made to use unit gravity sedimenta
tion to obtain proliferative and quiescent leukemic cells (6,
7). This technique is cumbersome and requires 4 to 5 hr.
We report the use of centrifugal elutriation as a simple,
rapid, and reproducible method for obtaining proliferative
plus quiescent subpopulations of leukemic cells. This
method permits the processing of up to 10@leukemic cells/
hr, obtained from a patient's peripheral blood or bone
marrow.
I This
research
was
supported
by
USPHS
Grants
CA-5834
and
CA-18420
from the National Cancer Institute and by the B. Hoffman Trust Fund.
2 The
abbreviations
used
are:
LI,
labeling
index;
EBSS,
Earle's
balanced
salt solution; TCJR,thymidine; ara-C, 1-fro-arabinofuranosylcytosine;
CR, 5-azacytidine.
Received May 4, 1977; accepted July 22. 1977.
3876
aza
was removed.
Lysis
was repeated once. Cells were washed twice with EBSS
(Grand Island Biological Co., Grand Island, N. V.), and
were suspended in EBSS.
Centrifugal Elutriatlon
A Beckman J-21B refrigerated centrifuge and a Beckman
JE-6 elutriator rotor (Beckman Instruments, Inc. , Palo Alto,
Calif.) were used in these studies. Two-step separation was
carried out.
Step 1. The specimen was introduced into the elutriator
rotor spinning at 2500 rpm with a flow rate of 29 mI/mm.
The effluent from the rotor was collected after an additional
190 ml of EBSS were pumped through the rotor at this flow
rate and was labeled Fraction 1. The flow rate of EBSS was
increased to 43 mI/mm and, after 210 ml were collected
(Fraction 2), the flow rate was increased to 90 mI/mm. The
centrifuge was stopped after a further 210 ml were collected
(Fraction 3). The separation chamber was removed from
the rotor, and the residual cells in the chamber were
recovered and designated as Pellet 1. The cells of Fractions
1 to 3 and Pellet 1 were recovered by centrifugation at 600
x g for 10 mm.
Step 2. The cells of Fraction 1 were then resuspended in
EBSS and reintroduced into the elutriator rotor (5000 rpm)
at a flow rate of 26 mI/mm. At incremental flow rates of 42,
57, 90, and 100 mI/mm, 210-mI sequential fractions were
collected and designated Fractions 4 to 8. Cells remaining
in the chamber were removed and designated Pellet 2.
CANCER RESEARCHVOL. 37
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Proliferative and Quiescent Leukemic Cells
Morphological Studies
Slides of the unseparated specimen (preelutriation) and
of each elutriator fraction were made with a cytocentrifuge.
They were stained with Wright-Giemsa, and differential
counting (200 cells) was carried out by a single investigator.
Cell size (50 cells) was determined with a Vickers AEI
image-splitting eyepiece.
Radioautographic Studies
Two million cells were suspended in 1 ml of NCTC 109
made 2% with fetal calf serum. Ten
@Ciof [3H]TdR
(Schwarz/Mann, Orangeburg, N. Y.) were added, and the
cells were incubated at 37°in a humidified CO2 incubator
for 30 mm. Ten ml of nonradioactive TdR solution (mg TdR
per ml of 0.9% NaCI solution) were added, and the cells
were washed 5 times with cold 0.9% NaCI solution. Slides
were made with a cytocentrifuge, washed to remove unin
corporated [3H]TdR, coated with Kodak NTB2 (Eastman
Kodak Co., Rochester, N. V.) emulsion, and placed in the
cold for 2 weeks. The radioautographs were developed
with a D-19 developer and stained with Wright-Giemsa. All
the radioautographs were read by a single observer, and
cells containing
@5
grains over their nuclei were considered
to be labeled.
Drug Metabolism
Each cell fraction including the prefraction was incubated
with 2.5 @Ci
of either [3H]-ara-C (Amersham/Searle Corp.,
Arlington Heights, Ill.) or [‘4C]-5-aza-CR
(Stanford Research
Institute, LaJolla, Calif. , through the courtesy of Dr. Robert
Engle, Division of Cancer Treatment, National Cancer Insti
tute, Bethesda, Md.) at 1.0 x 10@ M at 37°for 30 mm. Cells
were then washed twice with Roswell Park Memorial Insti
tute Medium 1640 (Grand Island Biological) containing 10%
fetal calf serum and 2% N-2-hydroxyethylpiperazine-N'-
2-ethanesulfonic acid Mopes (Grand Island Biological). Cells
free of extracellular label were then extracted with a mini
mum volume (100 @l/10@
cells) of 6% perchloric acid, the
precipitate was removed at 4°by centrifugation, and the
supernatant was then neutralized with 2 N KOH. Aliquots
were used to determine total radioactivity found in the
acid-soluble fraction . Acid-insoluble fractions were washed
with 6% perchloric acid, and the total radioactivity incorpo
rated into the macromolecules was determined by liquid
scintillation counting with 30 and 85% efficiency for 3H and
14C,respectively.
RESULTS
Table 1 gives the separation data for specimens from 3
representative patients: 2 bone marrow specimens and 1
specimen of peripheral blood. Cell populations with a
variety of LI's were obtained. In each case, fractions with
different-sized blast cells were obtained, and the LI's were
greatest in the fraction containing the largest cells. In
Patients 1 and 2, the blasts in Fraction 2 had the highest
LI, whereas in Patient 3 the Pellet 2 blasts had the highest
LI. Cell viability as determined by trypan blue dye exclusion
was >90% (usually 99%) in cell fractions containing >10@
cells. Total recovery of cells (the number of cells initially
introduced into the elutriator/sum of cell Fractions 2 to 8
and Pellets 1 and 2 after recovery) was approximately 59%.
This figure is representative of the 15 separations that we
have carried out to date (range, 19 to 100% with recovery
of <42% in only 2 patients). The larger the number of
cells separated, the higher is the cell recovery. The 2
specimens for which recovery was <42% consisted of <10@
cells at the start of separation.
Bone marrow specimens from 8 patients with acute
myelocytic leukemia and from 2 patients with acute lympho
cytic leukemia were studied. Peripheral blood specimens
from 5 of the 10 patients were also studied. Table 2 gives
the total number of bone marrow cells subjected to elutria
Table 1
patientPatient
Separationof
bone marrow specimens from2 patients and peripheral blood from 1
6No.
2Patient
1Patient
of
blasts
of
(sm)LIPreelutria cells%
of
AMLa
(sm)LINo.
x lO@
tion
Fraction
7.3 x l0@
Fraction 3
1.8 x 10.
PeIletl
1.3x10
74
20.8±0.8
0
ND
ND
4.7 x l0@
76
13.8 ±0.4
4
58
15.8 ±0.5
6
Fractions
4 to 5
Fraction 6
Fraction
Fraction
7
8
Pellet 21.1
x 10@
x 108
7.8 x l0@
51
2.8 x l073
77
a AML, acute myelocytic
b Average
blasts
of ALL
x 10'
19.1 ±3.8'
62
70
ND
1
Fraction 2
of
of
of
cells%
BM cell size
9 X 10
7.5 x 10'
ND
ND
ND
6
5
ND
ND
1 .3 x l0
ND
10.8 ±0.4
13.2 ±0.3
ND
ND13.9
1.8
19.6
1.3 x l0
4.8 x l0
51
67
ND
2.3 X l0
2.4 x 10@
4.5 X l0
75
41c
67
12.9 ±0.4
15.1 ±0.7
11.5 ±
48
2
22
ND
1 .3 x 10@
1.7 x l0@ 82
2
11.6 1.7 x l0
82
17.2 ±0.6
ND
ND
ND
18.8 ±0.634.8ND4.5 3 x l0@71 43
leukemia;
BM, bone marrow;
72
82
71
64
17.9
44.1
18
64
ND
ND
±0.4
13.4 ±0.4
17 ±0.5
20.3 ±0.8
ND
xlOt
cell
blasts
of cells% of PBAML
size
BM cell
size (sm)LINo.
ALL, acute lymphocytic
X l0
xlO4
ND
ND
ND10' 1.1 x l0@65
leukemia;
PB, peripheral
41d12.1
blood;
12
13.6
12.2
13
±0.3
±0.5
±0.4
±0.5
ND
0.2
1.6
ND
ND
ND
10.8 ±0.3
0
12.4 ±0.4
ND
ND
ND
15.3 ±0.60.4 7.8
ND, not done.
± SE.
C Promyelocytes
constituted
36%
of
cell
population.
d Promyelocytes
constituted
20%
of
cell
population.
NOVEMBER1977
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3877
H. D. Preisler et al.
Table 2
centrifugalelutriation% Separationof leukemic bone marrow by
of blastsCell
size―
(.tm)LINo.
cellselu
of
Pro
Pro
Pa
tria
High
Low
Preelutria
elutri
High
Preelutria
Low
tienttionestesttionHighestLowestationestesttionHighestLowest1737076l9.l±O.4b20.3±0.813.8±0.434.8642l06
xlOT4.7x10391929717.9
±0.6―18.2
10'4706584l7.5±[email protected]±0.615.1±0.4412.30.47.4xlO'l.9xlO'3.5x10'5929910017.7
x 10' ‘
5.7x
±0.417.5 ±0.38.44233.2
x l02.6
±0.4c18.3 ±0.316.1
10679969112.8
10780888512.2
±0.2―16.5
±0.312.9
±0.4b13.6
±0.412.4
±0.5―15.2±0.513.5
±0.7b20.1
±0.716
±0.4―15.1
±0.710.8
±0.3―17.2 ±0.513.8
10'884779714.1
10'983888817.8
10'271418213.4
10'1094829313.4
a Average
±0.44.660.21.8
±0.45.213.401.3
x [email protected] x 10'6.9
x l03.3
x l01.3
±0.35.37.901.3
x l01.1
±0.35.320.21.0107
±0.413.758.95.83.5
±0.413.9441.84.5
±0.512.754.797
x 10'6.7
x
x 10'6.7
x
x
x 10'1.5
x l03
x 10'2.4 x 10'1.6 x
x 10'1.5
x 10'3.9
X 10'
± SE.
b Acute
myelocytic
C Acute
promyelocytic
d Acute
lymphocytic
leukemia.
leukemia.
leukemia.
tion as well as the average cell size and LI for each patient
studied. The same data is given for the specific cell frac
tions that had the highest and lowest LI of each specimen
subjected to elutriation.
The average sizes of the unseparated marrow blasts
ranged from 12.2 to 19.1 @m,with LI's varying from 4 to
34.8%. There was no clear relationship between the average
cell size in the unseparated specimen and the LI. In every
case elutriation provided separate subpopulations of cells
for which the LI's both exceeded and were less than that of
the unseparated marrow. By consideration of the marrow
specimens, the LI's of 6 of the 10 fractions with the highest
LI's exceeded 20%, and in 5 the LI's of the most active
fractions exceeded 40%. By contrast, the LI's of the frac
tions with the lowest LI's were one-sixth to one-thirtieth
(within each patient cell subpopulation) those of the most
active fractions. In 7 of the patients, there was a 10- to 30fold difference in LI's between the most active and the
least active cell populations. The fractions with the highest
LI's always contained larger blasts than did the fractions
with the lowest LI's (1 to 5 @tm
difference in mean diameter).
In one case the fractions
with the highest
LI's (Patient 2)
had substantially less blasts than did the other fractions.
Centrifugal elutriation was used to separate 5 peripheral
blood specimens into proliferating and quiescent popula
tions (Table 3). In 3 of the 5 specimens, the LI's of the most
active fractions varied between 3.6 and 7.8%. In the speci
mens of the other 2 patients, the maximum LI's obtained
were 28.1 and 36.7%, respectively. In each case the speci
men with the highest LI had @64%
blast cells. The relation
ship between the differences in average cell size and LI in
relation to the marrow specimens was also found. In each
case the size and LI of the subpopulations of peripheral
blood blasts were less than the corresponding marrow
fractions from the same patient.
Uptake of ara-C and aza-CR
The uptake of ara-C and aza-CR by unseparated leukemic
cells and by elutriator-separated subpopulations has been
3878
x
x
studied in the specimens of 5 patients (3 marrow specimens
and 2 specimens of peripheral blood). Table 4 gives the
data obtained from 2 representative studies. In Patient 9,
Fraction 2, which has the highest LI, has the lowest uptake
of ara-C. For Patient 8, Fraction 7 has a 20-fold higher LI
than does Fraction 6 and does in fact take up more ara-C
and aza-CR. However, Fraction 1, which has a much lower
LI than does Fraction
7, takes up an equivalent
amount of
ara-C but substantially less aza-CA. Hence, it appears that
there is no consistent relationship between the amount of
ara-C and aza-CR taken up by the cells and the LI of the
cell population. Furthermore, the uptake of each drug
appears to vary independently from each other.
DISCUSSION
Centrifugal elutriation provides a simple and rapid means
for separation of leukemic cells into subpopulations with
different
LI's. Using this technique,
we have been able to
obtain, from the same patient, subpopulations of cells for
which the LI's differ by as much as 30-fold. In 8 of the 15
specimens studied, we have obtained subpopulations for
which the LI's exceeded 20%. In 5 of the 10 marrow
specimens studied, we obtained'fractions
for which the
LI's exceeded 40%. This is especially significant since the
reported LI for actively proliferating leukemic marrow spec
imens has been estimated to be 50% (5). Similarly, we
obtained subpopulations for which the LI's were <3% in 11
patients (@1% in 8 patients). Hence, from one-half of the
patients studied, we obtained subpopulations of cells that
were highly purified for both proliferative and quiescent
leukemic cells.
The specific elutriator fraction containing the most ac
tively proliferating subpopulation of cells varied from pa
tient to patient. The higher the initial Ll, the greater
was the likelihood of obtaining a subpopulation with a very
high LI. For the 5 patients whose marrow LI's were @8%,
subpopulations of cells were obtained whose LI's varied
CANCER RESEARCHVOL. 37
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Proliferative and Quiescent Leukemic Cells
Table 3
elutriationPatient%
Separation of leukemic penpheral bloodcells
blasts
Pre
elu
tria
HighLow
tionof
tionHighestLowest5
est
estCell
6
10
41
28
64
cellsPreelutria
elutri
tionHighestLowestPreelutria
±0.7b
±0.4
12.1 ±0.4c 15.3 ±0.6
13.9±O.4d 15.1 ±0.3
ll.9±0.2'@ 12.3
7
65
91
78
957
7055 5955 [email protected]
a Average
bycentrifugal
size―(nm)Pro
51
97
78
15.715.6
of
High
ationLI estLowestNo.
x 10'
±0.3
7.8
10.8 ±0.3
12.2 ±0.4
11.8±0.3
0.4
14.52.4
1.66.2 28.13.4 0.25.4
1
0.3
0
10'
x 10'
1.1 x 10'
x 10'
1.3 x 10'
1.5 x 10'
3.9 x 10'
36.7
0.9
1.1 x 10
3.6
0.2
9.6xlO'
5.4x10'
l.2x10'
1 x 107.9 1.6x 10'2.43.3x 10'
±S.E.
b Acute
promyelocytes.
C Acute
myelocytic
d Acute
lymphocytic
leukemia.
leukemia.
Table 4
Drug uptake by proliferative and quiescent leukemic blast cells
Patient 9Uptake
8Patient
(nmoles/10'Uptake(nmoles/10'%of
blastsLIcells)%of
aza-CRara-Caza-CRPreelutriation845.37.0
blastsLIcells)ara-C
55.78313.72.692.3Fraction
18833.9
.2807.62.964.8Fraction
61
28858.90.8176.4Fraction
6971
107.9Fraction77720.24.1
.01
.2
251.3Pellet
26543.13.594.1
between 42 and 64%. By contrast, for the 5 patients whose
unseparated marrow LI's were <8% (4 to 5.3%), the LI of
the most actively proliferating fraction varied beween 6 and
that
this
resulted
from
the
big differences
in cell
size
between very small and very large blasts. Hence, at a rotor
speed and flow rate at which the smallest and largest cells
are simultaneously retained in the chamber, the flow rate
20.2% (for the 4 patients, <13%).
of the suspending media provides insufficient force to keep
Although elutriator separation is determined to a signifi
cant degree by differences in cell size, density factors also the largest blasts in suspension.
Centrifugal elutriation provides a rapid means of separat
play a role. Presently, marrow or peripheral blood speci
ing relatively pure cell populations into subpopulations
mens containing significant numbers of nucleated erythro
that differ in cell size. Although in some cases the number
cytes are unsuitable for separation. In such specimens the
of cells obtained in the actively proliferating cells is rela
elutriation fractions containing large leukemic blast cells
tively low (1 to 2 x 10'), even these cell subpopulations
are heavily contaminated with smaller nucleated erythroid
cells. Hence, for biochemical and/or drug uptake studies
can be used for some biochemical and most tissue culture
in which a high degree of cell purity is essential, prior
studies. Our initial biochemical studies have yielded an
removal of such contaminating elements is necessary. We unexpected observation; i.e., the uptake of ara-C appears
require
thata specimen be composed ofatleast70% blasts to be unrelated
to the proliferative
rateof the cellpopula
to be suitable for elutriation. Under these conditions (using
tion. This was somewhat of a surprise since ara-C selec
a specimen containing predominantly one type of cell), we tively kills cells in S phase. We are currently determining
have found that separation by elutriation is determined
the retention time of 1-fJ-D-arabinofuranosylcytosine 5'-tri
primarily by differences in cell size.
phosphate by the actively proliferating and quiescent leu
Our initial studies also demonstrate that it is necessary
kemic cells and are attempting to determine whether ara-C
to remove erythrocytes from specimens. Since the separa
taken up by quiescent leukemic cells damages these cells.
tion chamber can efficiently hold only 10' cells each time,
It may be that quiescent cells that take up ara-C are not
erythrocytes should not be present if the intent is to sepa
immediately damaged but are killed when they attempt to
rate 10' nucleated cells. Using leukemic cells we have
synthesize DNA. If this is the case, then uptake and reten
found that it is necessary to carry out the elutriation in 2 tion of ara-C by quiescent leukemic cells may be a determin
steps. On the 1st run we obtain Fractions 1 to 3 and Pellet
ing factor in the success or failure of the treatment of
1 . We subsequently separate Fraction 1 into subpopulations
acute leukemia with ara-C. Alternatively, the retention time
by another elutriator run. Prior to initiating this 2-step
of 1-f3-D-arabinofuranosylcytosine 5'-triphosphate by quies
procedure, we were plagued by the formation of a large
cent cells may be of insufficient duration to affect the
pellet of cells within the separation chamber. We believe
survival of the quiescent leukemic cells.
NOVEMBER 1977
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3879
H. D. Preisler et al.
ACKNOWLEDGMENTS
@
Theauthorsthank G. Christoff, EdytheTaylor,Jill Rubenstein,and Carol
Wrzosek for their excellent technical assistance.
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CANCER RESEARCHVOL. 37
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1977 American Association for Cancer Research.
Separation of Leukemic Cells into Proliferative and Quiescent
Subpopulations by Centrifugal Elutriation
Harvey D. Preisler, Irene Walczak, Joyce Renick, et al.
Cancer Res 1977;37:3876-3880.
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