A SIMPLE METHOD FOR THE SEPARATION OF LEUKOCYTES
FROM WHOLE BLOOD
EDMUND KLEIN, M.D., SANDRO ERIDANT, ISAAC DJERASSI, M.D., AND
ROBERT RESNICK, M.D.
Children's Cancer Research Foundation, and the Division of Laboratories and Research of The Children's
Medical Center, Boston, Massachusetts
The method described below facilitates the
separation of biologically active blood cells from
whole blood and plasma without the need of
specialized equipment.
MATERIALS
Glass syringes.
Venipuncture needles.
Erlenmeyer flask.
Perforable stopper.
Biuret stand and clamps.
Sedimenting agent for red blood cells (RBC).
Anticoagulant.
Blood.
METHOD
All surfaces with which leukocytes come into
contact are coated with hemorepellent agents.
Glass surfaces are coated with Silicone* and
Received, November 9, 1957; revision received,
January 7, 195S; accepted for publication January
16.
Dr. Klein is Research Associate, The Children's
Medical Center and The Children's Cancer Research Foundation; Clinical and Research Fellow,
Department of Dermatology, Harvard Medical
School at the Massachusetts General Hospital.
Dr. Eridani is Research Assistant in Pathology,
The Children's Medical Center and The Children's
Cancer Research Foundation, Boston, Mass.
Dr. Djerassi is Research Associate, The Children's Medical Center and The Children's Cancer
Research Foundation; Assistant in Pathology,
Harvard Medical School, Boston, Mass.
Dr. Resnick is Research Assistant in Pathology,
The Children's Medical Center and The Children's Cancer Research Foundation, Boston,Mass.
This work was supported in part by a grant
from "Aid for Cancer Research" and from grant
CY3335, U.S. Public Health Service, Department
of Health, Education, and Welfare, National Institutes of Health.
Dr. Klein was supported b.y a Fellowship in
the Medical Sciences, National Research Council,
Washington, D. C.
* General Electric No. 9977.
550
metal surfaces with Arquad,t according to the
method of Tullis and Rochow.10
After the blood is drawn into syringe A (Fig.
1), it is mixed with the appropriate amounts of
sedimenting agent and anticoagulant. Dextran,
1 part of a 10 per cent solution in physiologic
saline, was added to 9 parts of blood to accelerate
the sedimentation of red cells. The anticoagulant
used was % ml. of a 5 per cent solution of the
disodium salt of ethylenediamine tetra-a'cetic
acid (Sequestrene) per 10 ml. of blood. The syringe
was then placed in the position shown in Figure 1.
The red cell-depleted plasma is transferred to
the attached flask B, through the curved needle
C, which perforates the stopper D. The purpose
of the second syringe, E, is 2-fold: first, to provide
a vent for the flask, B; and second, to provide a
means of aspirating the plasma-cell suspension
from the flask, B.
The time required for the sedimentation of red
cells depends on the ratio of white blood cells
(WBC) to red blood cells (RBC) desired. After 30
min., a ratio of 5 WBC to 1 RBC is obtainable
with approximately 70 per cent recovery of
leukocytes. For higher WBC:RBC ratios, correspondingly longer sedimentation is required and
lower WBC yields are obtained. The highest
WBC:RBC ratio obtained was 50 WBC to 1
RBC.
In order to increase the ratio of leukocytes to
platelets, the plasma containing both these
formed elements is aspirated into syringe, E,
which is then inverted. The white cells settle by
gravity within 60 to 120 min., leaving approximately SO per cent of the platelets in suspension.
Further removal of platelets can be effected by
centrifugation at 600 g for 5 min., which results in
a leukocyte layer containing less than 20 per
cent of the original platelet number. This leukocyte sediment represents between 40 to 50 per
cent of the original WBC complement (i.e., 30
t Armour and Company, Chicago, Illinois, No.
75, Lot 701.
June 1958
551
SEPARATION OF LEUKOCYTES
FIG. 1. Apparatus used for separation of leukocytes from whole blood.
ml. of blood with a leukocyte count of 8400
W.IJC per cu. mm. yielded a total of 115,000,000
WBC). The differential count shifts during this
procedure, resulting in a relative increase in the
number of the polymorphonuclear elements and
monocytes, and a corresponding decrease in the
number of lymphocytes (Table 1).
The white cell layer, which is obtained by a
recently developed method5 for separating blood
elements, has been used as starting material
for further separation by this procedure. A similar
principle has been employed by Buckley and
Gibson.3 Further separation of white cells can be
effected by a number of more specialized procedures, i. 2. 4. 9. U
The leukocyte concentrates prepared by this
method have been satisfactory without further
purification in a number of studies. The effects
of varying concentrations of divalent cations
(calcium, magnesium, barium, strontium, copper,
and zinc) on polymorphonuclear phagocytosis was
TABLE 1
DIFFERENTIAL WHITE BLOOD CELL COUNTS OF
SPECIMEN PRIOR TO AND AFTER SEPARATION
OF W H I T E
BLOOD
N e u t r o - Eosinophil es
philes
CELLS
Basophil es
Monocytes
per cent per cent per cent per cent
Prior to separation
After
separation
Lymphocytes
per cent
64
2
1
3
30
76
5
1
6
12
552
K b ELY ET
investigated in these preparations. 6 I t was found
t h a t optimal concentrations of these ions increased t h e phagocytic index from 5 t o 10 p e r
cent (controls without divalent cations) t o 80
to 100 per cent. These W B C preparations were
also used in this laboratory as antigens for t h e
production of heterologous antileukocyte antibodies. 8 Low concentrations of antiserum produced agglutination of W B C ; intermediary
concentrations inhibited phagocytosis, b u t did
not produce agglutination (pro-zone phenomenon) ; high concentrations of antiserum resulted
in lysis of W B C . White cell preparations obtained
as described here have been used b y Moloney
and K e n n y for histochemical studies. 7 T h e
method h a s also been used for collecting white
cells for differential counts in agranulocytosis.
SUMMARIO IN INTERLINGUA
E s describite un methodo que facilita le separation d e biologicamcnte active cellulas d e sanguine
a b sanguine integre e plasma sin le requirimento
de un a p p a r a t u r a o altere dispositivosspecialisate.
Le methodo h a essite usate con bon successo in
studios histochimic e in colliger leucocytos pro
numerationes differential in agranulocytosis.
REFERENCES
1. A G B A N O F F , B . W., V A L L E E , B . L., AND W A U G H ,
D. F . : Centrifugal subfractiomition of
polymorphonuclear leukocytes,
lympho-
Vol. 29
AL.
cytes and crvthrocvt.cs.
839, 1954.
Blood, 9: S04-
2. B R A U N S T E I N E R , H., PAKESCH, F . , AND V E T T E R ,
H . : Isolierung und Anreicherung Funktionstiichtiger Leukocyten und Thrombocyte!!
mittels Polyvinyl-Pvrrolidin. Acta haemal,., 8: 304-308,'.1952.
3. B U C K L E Y , E. S., AND G I B S O N , J . G., I I : Un-
published d a t a , 1950.
4. H A G E R M A N , D . D . , AND D E N N Y , G. H . , J R . :
The cholinesterase activity of human leukocytes. U.S. Army Scientific Report no. 7,
1956.
5. K L E I N , E . ,
ARNOLD, P . , E A R L , R. T . , AND
W A K E , E . A.: A practical method for t h e
aseptic preparation of human platelet concentrates without loss of other blood elements. New England J . Med., 254: 11321133,4956.
6. K L E I N , , E . , AND E R I D A N I , S.: T h e effect
of
bivalent cations on polymorphonuclear
phagocytosis (In p r e p a r a t i o n ) .
7. M O L O N E Y , W. C , AND K E N N Y , J . J . : L e u k o c y t e
alkaline p h o s p h a t a s t e ; behavior during
prolonged incubation and infection in
normal and leukemic leucocytes. Blood,
12: 295-302, 1957.
8. R E S N I C K , R., AND K L E I N , E . : T h e inhibition of
polymorphonuclear phagocytosis by antibodies against white blood
cells. J.
Lab and Clin. Med. (in press).
9. T U L L I S , J . L . : Separation and purification of
leukocytes a n d platelets. Blood, 7: 8 9 1 896, 1952.
10. T U L L I S , J .
wettable
1952.
L.,
AND R O C H O W , E .
surfaces.
Blood,
7:
G.:
Non-
S50-S53,
11. V A L L E E , B . L., H U G H E S , W., J R . , AND G I B S O N ,
J . G., I I . : A method for t h e separation of
leukocytes from whole blood by flotation on
serum albumin. Blood, Special Issue, no.
1, p . 82-87, 1947.