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Monocyte purification with counterflow centrifugation monitored by continuous flow cytometry
Journal of Immunological Methods, 47 (1981) 31--38 Elsevier/North-Holland Biomedical Press
31
MONOCYTE PURIFICATION WITH COUNTERFLOW C E N T R I F U G A T I O N M O N I T O R E D BY C O N T I N U O U S F L O W CYTOMETRY '
P.H.M. DE MULDER, J.M.C. WESSELS, D.A. ROSENBRAND, J.B.J.M. SMEULDERS, D.J.Th. WAGENER and C. HAANEN Division of Hematology, Department of Internal Medicine, University Hospital St. Radboud, Geert Grooteplein Zuid 16, 6525 GA Nijmegen, The Netherlands
(Received 29 March 1981, accepted 8 July 1981)
Continuous monitoring of cell light scatter during counterflow centrifugation of a mononuclear cell suspension allows counting and size recognition of the cell types elutriated. With this method an optimal separation point between monocytes and lymphocytes, determined for each individual donor, may be established. With a constant flow of 15 ml/min this separation point is found at centrifugal velocities ranging from 2348 to 2444 rpm (n = 10). From 50 ml venous blood, 84.1% + 4.1% (15.7 + 8.6 × 106 ) of all elutriated monocytes, with a purity of 92.4% + 1.4%, is collected in a volume of 50 + 1 ml. In the same run, 92% + 4.3% of the lymphocytes is gathered in one fraction with a purity of 98.9% + 0.7%. After counterflow centrifugation, 91.6 + 10.5% of the cells loaded is recovered; viability exceeds 98%. INTRODUCTION O p t i m a l s t u d y o f i m m u n o l o g i c a l and n o n - i m m u n o l o g i c a l f u n c t i o n s o f m o n o c y t e s requires purified m o n o c y t e suspensions. C o n v e n t i o n a l isolation t e c h n i q u e s are based u p o n either t h e i r a d h e r e n t p r o p e r t i e s (Koller et al., 1 9 7 3 ; A c k e r m a n and Douglas, 1 9 7 8 ; R i n e h a r t et al., 1 9 7 8 ) or d e n s i t y ( L o o s et al., 1 9 7 6 ; N a t h a n s o n et al., 1 9 7 7 ; U l m e r a n d Flad, 1 9 7 9 ) . M o n o c y t e isolat i o n b y c o u n t e r f l o w c e n t r i f u g a t i o n (elutriation) has r e c e n t l y been s h o w n t o give high p u r i t y , g o o d r e c o v e r y , and excellent viability a n d f u n c t i o n (Sanderson et al., 1 9 7 7 ; Weiner and Sha, 1 9 8 0 ; C o n t r e r a s et al., 1 9 8 0 ; L i o n e t t i et al., 1 9 8 0 ) . S e p a r a t i o n is based m a i n l y o n d i f f e r e n c e s in cell size a n d to a m u c h lesser e x t e n t o n d e n s i t y . M e t i c u l o u s r e g u l a t i o n o f flow, centrifugal f o r c e and t e m p e r a t u r e are essential t o achieve o p t i m a l p u r i f i c a t i o n . A f t e r relatively ' b l i n d ' e l u t r i a t i o n in f r a c t i o n s , screening o f t h e e f f l u e n t is usually p e r f o r m e d b y m e a n s o f a C o u l t e r c o u n t e r . In this p a p e r a n e w m e t h o d is described w h e r e b y c o n t i n u o u s m o n i t o r i n g o f t h e e l u t r i a t o r o u t p u t is o b t a i n e d , 1This study was supported by the Queen Wilhelmina Foundation, The Netherlands Organisation against Cancer (KWF/NOK). 0022-1759/81/0000--0000/$02.75 © 1981 Elsevier/North-Holland Biomedical Press
32 allowing simultaneous counting and size quantification. In this way it is possible to determine the optimal separation point between the different mononuclear cell populations in each elutriator procedure, resulting in improved efficiency of counterflow centrifugation. MATERIALS AND METHODS
Collection of blood Approximately 5 0 m l venous blood from healthy volunteers, anticoagulated with 6 IU/ml preservative-free heparin, is obtained b y venepuncture. An aliquot is taken for total and differential cell counting. Preparation of the mononuclear cell suspension (MNL ) The blood is immediately diluted 3-fold with calcium- and magnesium-free Hank's buffered balanced salt solution (HBBS), supplemented with 100 mg di-K-EDTA/1, final pH 7.3, 295 mosM/kg. Thirty-five ml samples are layered on 15 ml 1.077 g/ml Ficoll-Isopaque (pH 6.6, 272 mosM/kg). After centrifugation (400 X g; 30 min; 20°C) and removal of the supernatant, the interphase is collected, diluted with HBBS + 0.4% bovine serum albumin w/v (BSA), washed once ( 6 0 0 X g , 1 5 m i n , 4°C) and resuspended in 5 - - 8 m l HBBS + 0.4% BSA. A fixed volume is taken for counting and differentiation. The remaining cells are introduced into the elutriatior. Elu tria tion Elutriation system. This consists of a J2-21C centrifuge equipped with the JE-6 elutriator rotor and standard separation chamber (Beckman Instruments, Palo Alto, CA, U.S.A.). The flow is maintained with a roller pump (Masterflex4~ole-Parmer Instruments, Chicago, IL, U.S.A.) and pulsations are flattened b y a pulse suppressor chamber. Medium temperature is maintained at 10°C. The J2-21C centrifuge is modified by installing a fine scaled speed selector (one scale unit: 2.6 -+ 0.1 rpm) and replacing the C102 condenser (0.022 UF) b y a condenser of 47 pF; this results in improved stability of rotor speed. R p m measurement was altered by using a high resolution counter (120 HMZ pH 6667 Philips, Eindhoven, The Netherlands). The elutriator effluent is collected in a small d o w n f l o w cabinet. HBBS + 0.4% BSA w/v is used as elutriation medium. Cell recognition device (Fig. 1). Continuous sampling of the elutriator o u t p u t is established by means of a T-drain. This sample stream (a, 0.2 ml/ min) mantled in a sheath stream (b, 1.4 ml/min), is continuously analyzed for cell number and cell light scatter by the electro-optical peroxidase unit of the Hemalog-D (Technicon Instruments Corp., Tarrytown, U.S.A.). This unit, dismantled from the Hemalog-D, consists o f a flow cell with optical assembly for focusing the light from a tungsten halogen light source and an absorption and scatter photodiode. The light scatter signals are accumulated and displayed on an ND-100 multichannel analyzer (Nuclear Data Inc., Schaumberg,
33
1
2
3
Fig. 1. Schematic representation of elutriator rotor (1) and the cell recognition device (2), consisting of: (a) sample stream directly derived from elutriator effluent; (b)sheath flow; (c) roller p u m p ; (d) electro-optical unit; (e) ND-100 multichannel analyzer. Collection of elutriator o u t p u t (3).
IL, U.S.A.). The resulting histogram (Fig. 2) indicates the number o f cells as a function o f cell size (scatter). When a new cluster o f cells with a markedly different scatter signal is elutriated, a change in the histogram appears on the monitor. At any time the profile can be erased or stored in a m e m o r y , enabling direct comparison o f elutriated cell populations. Elutriation technique. Prior to introduction o f the cell suspension, entrapped air is removed, rotor speed adjusted to 2500 rpm, and medium flow established at 15 ml/min under control o f a flow-meter (Sho-rate flowmeter t y p e No. R-2-25-B Brooks Instruments Inc.). By means o f an infusor, the cell sample is introduced directly into the tubing line at a flow-rate of 0.3 ml/min. During this period m o n o c y t e s and larger l y m p h o c y t e s remain in the separation chamber, whereas smaller lymphocytes, erythrocytes and t h r o m b o c y t e s are eluted. When introduction o f the cells is completed the rotor speed is lowered 26 -+ 1 rpm every 2 min. The decrease o f centrifugal force results in an o u t f l o w o f l y m p h o c y t e s with increasing diameter, while the n u m b e r of cells eluted per time unit diminishes. At a certain rotor speed, indicated as the separation point, cells with a distinct scatter signal (monocytes) leave the elutriator (Fig. 2, profile 2). Since l y m p h o c y t e s are still being washed out, a double peaked histogram is seen, representing the t w o cell populations. The rotor speed is then immediately increased 15 rpm in order to wash o u t the remaining l y m p h o c y t e s and to retain the monocytes. Collection o f fraction 2 is started. When the number o f cells leaving the separation chamber falls below 100 cells/sec, the rotor is stopped and the effluent collected in one 50 ml t u b e (fraction 3). Immediately thereafter a 'rest' fraction is gathered (fraction 4). After washing with HBBS + 0.4% BSA (600 X g, 15 min, 4°C} the cells are resuspended and further analysis is performed. The whole procedure can be carried o u t within 1 h.
34
PRIOR
TO
ELUTRIATION
500
of
j, i
,
i
700
DURING
AFTER
500
0 0
II
500
| <
z
~o
a
6O0 CHANNEL SCATTER
60O
NUMBER (SIZE~
Fig. 2. Histograms as seen on the monitor of the ND-100 multiehannel analyzer. Prior to elutriation: the mononuclear cell suspension. During elutriation: profile 1 represents lymphocytes, profile 2 represents the mixed population and profile 3 monocytes. After elutriation: scatter analysis of the entire lymphocyte, mixed, and monocyte population.
35
Cell counting and differentiation Cell number in the starting material, MNL and elutriated fractions is measured with a Coulter counter. Cytocentrifuge preparations o f the MNL and elutriated fractions are made according to Talstad and Gundersen (1979) and stained with May-Griinwald-Giemsa and non-specific esterase, respectively (Yam et al., 1971; Lawrence and Grossman, 1979). Differential counting is performed on 400 cells. Cell viability Cell viability is assessed b y t w o methods. (1) Cells suspended in a concentration of 3--5 × 10S/ml are mixed with 20 #l fluorescein di-acetate/ml (5 mg FDA/ml acetone) and 20 pl ethidium bromide/ml (0.0025 mg EB/ NaC1 35.8 mg/ml and Tris 12.1 mg/ml, pH 7.5). Viable cells are FDA-positive (green cytoplasmic fluorescence; Celada and Rotman, 1967) and dead cells ethidium bromide-positive (red DNA fluorescence). (2) Exclusion o f trypan blue b y viable cells. RESULTS If the rotor speed is continuously decreased and the flow-rate maintained constant, cells with a gradually increasing diameter will leave the elutriator. With the effluent monitored by the cell recognition device measuring light scatter to analyse cell number and size, it is possible to record the distribution of these parameters in a mononuclear cell suspension (Fig. 3). In this particular experiment the monitoring was started after elutriation of the t h r o m b o c y t e s . The first and highest peak represents the lymphocytes, the second contains the monocytes. This form of cell scatter registration allows recognition of the different cells during elutriation. The possibility o f immediate identification of cell t y p e combined with low elutriator o u t p u t between given cell populations is the basis of this separation method. The histograms registered during elutriation are comparable with those of the final separated fractions (Fig. 2). The results of 10 runs are listed in Table 1. Fraction 3 consists of elutriated m o n o c y t e s of high purity with only slight admixture of l y m p h o c y t e s (6.5 + 1.6%), some basophils, and very few segmented neutrophils. The majority of the l y m p h o c y t e s is present in the first fraction with a purity of 98.9 -+ 0.7%, with only 1.1 + 0.6% monocytes. In the second fraction a mixed population is found: 2.6 + 4.3% of the lymphocytes and 6.2 -+ 2.8% of the elutriated monocytes. Finally a less pure m o n o c y t e fraction (80.8 -+ 5.6%) with 17.8 +- 4.5% l y m p h o c y t e s is gathered, representing only 5.5 -+ 3.1 and 0.5 + 0.3% respectively of the total eluted cell number. The rotor speed (rpm) at which, for the first time, two cell populations are seen, is defined as the separation point. This is found at 2392 -+ 39.5 rpm with a range of 2348--2444 rpm. Viability o f the elutriated cells is 97.7 + 1.1% as determined b y E B / F D A and 98.6 + 0.5% by trypan blue.
36
SCATTER
{SIZE)
Fig. 3. Relationship between cell number and light scatter signal of 44 consecutively elutriated fractions. Each of the 44 histograms represents the number and the corresponding light scatter of the cells elutriated in 150 sec. With the same interval the rotor speed was diminished 26 ± 1 rpm, while the flow remained constant at 15 ml/min. Fraction 1 is comparable to histograms 1--29; fraction 2 to histograms 29--32; fraction 3 and 4 to histograms 32--44. A f t e r e l u t r i a t i o n , 9 1 . 6 -+ 1 0 . 5 % o f t h e l o a d e d c e l l s a r e r e c o v e r e d . B y t h e m e t h o d d e s c r i b e d , 8 8 . 5 -+ 1 7 % o f t h e m o n o c y t e s a n d 6 4 . 8 + 9 % o f t h e l y m p h o c y t e s p r e s e n t in t h e p e r i p h e r a l b l o o d s a m p l e a r e r e c o v e r e d i n t h e e l u t r i a t o r e f f l u e n t , o f w h i c h 7 2 % a n d 6 0 % r e s p e c t i v e l y a r e f o u n d in t h e p u r e s t
TABLE 1 Purity and yield of lymphocytes and monocytes in the 4 elutriated fractions (mean % ± S.D.). Yield is expressed as the relative distribution of lymphocytes and monocytes over the 4 fractions; purity as the composition within each fraction. Fraction
1
2 3 4
Monocytes
Lymphocytes
Purity (%)
Yield (%)
Purity (%)
Yield (%)
4 . 1 ± 1.8 23.9±13.8 9 2 . 4 ± 1.4 8 0 . 8 ± 5.6
1.1±0.6 6.2±2.8 84.1±4.1 5.5±3.1
9 8 . 9 ± 0.7 75.4±13.7 6 . 5 ± 1.7 1 7 . 8 ± 4.5
92 ± 4 . 3 6 ±4.3 1.5±0.6 0.5±0.3
37 fractions. The absolute n u m b e r of m o n o c y t e s and l y m p h o c y t e s found in those fractions is (15.7 ± 8.6) X 106 and {63.9 ± 20) X 106 respectively. DISCUSSION Purified m o n o c y t e s are needed for study of their immunological and nonimmunological functions. Isolation on the basis of functional properties means selection and possible changes in cellular metabolism induced b y the isolation technique (Bodel et al., 1977). Counterflow centrifugation avoids this and provides an excellent m e t h o d for cell separation. Sanderson et al. (1977) were the first to report considerable purification o f a small quantity of m o n o c y t e s by elutriation. Weiner and Sha (1980), Lionetti et al. (1980), Contreras et al. (1980) and Figdor et al. (1981) described the isolation o f large quantities. A disadvantage o f the technique, however, is the relatively blind separation in fractions obtained at flow-rates and rotor speeds based upon results o f previous experiments. The use of a cell recognition device, continuously measuring elutriator o u t p u t , overcomes this problem. Firstly, one can actually see when a cohort of cells is collected. In our experiments fraction 1 is collected in 456 ± 36 ml, fraction 2 in 116 ± 56 ml and fractions 3 and 4 each in 50 -+ 1 ml. Secondly and even more important, the technique allows immediate recognition o f the eluted cell type. Contreras et al. (1980) stated that the elutriation characteristics of each donor's mononuclear cell suspension are different, and each run has to be tailored individually for each donor. The relatively wide range of rpm (2348--2444) found as the separation point in o u r study confirms this and corroborates the advantage of a separation m e t h o d involving direct visualization and recognition. On the other hand Fogelman et al. (1979) and Figdor et al. (1981) d o c u m e n t e d highly reproducible m o n o c y t e isolation from run to run w i t h o u t individual adjustment. In addition to the variability of flow, rotor speed and temperature already mentioned, which, if not standardized for each run, influence the elutriation characteristics considerably; biological spread in cell volume may account for the variation in separation point observed in the present study and m a y be of importance when dealing with patients' mononuclear cell suspensions. Using the isolation technique described, we achieved excellent purification o f m o n o c y t e s and l y m p h o c y t e s with good recovery and viability. Moreover the m o n o c y t e s are gathered in a small effluent volume (50 ml). The results reported here with a relatively small (50 ml) aliquot of peripheral venous blood as the source of the MNL suspensions and actual identification o f the eluted cell type, support the potential application of this purification technique in the study o f m o n o c y t e function in patients. Preliminary results on blood from patients with Hodgkin's disease, non-Hodgkin lymphomas, and infectious mononucleosis are essentially similar to those reported here.
38 ACKNOWLEDGEMENTS
We are indebted to A. Plas and L. Coenen for their skilful assistance with the installation and adjustement of the elutriation equipment and Technicon Instruments, Rotterdam, The Netherlands, for kindly supplying the peroxidase unit. We acknowledge J. Van Egmond's help with the computer operation. REFERENCES Ackerman, S.K. and S.D. Douglas, 1978, J. Immunol. 120, 1372. Bodel, P.T., B.A. Nichols and D.F. Baintaon, 1977, J. Exp. Med. 145,264. Celada, F. and B. Rotman, 1967, Proc. Nail. Acad. Sci. U.S.A. 57,630. Contreras, T.J., J.F. Jemionek, H.C. Stevenson, V.M. Hartwig and A.S. Fauci, 1980, Cell. Immunol. 54,215. Figdor, C.G., W.S. Bont, J.E. De Vries and W.L. Van Es, 1981, J. Immunol. Methods 40, 277. Fogelman, A.M., J. Seager, M. Hokom and P.A. Edwards, 1979, J. Lipid Res. 20,379. KoUer, C., G. Kind, P. Hurtibise, A. Sagone and A. Lobuglio, 1973, J. Immunol. 111, 1610. Lawrence, C. and R. Grossman, 1979, Stain Technol. 54,321. Lionetti, F.J., S.M. Hunt and C.R. Valeri, 1980, in: Methods of Cell Separation, Vol. 5, ed. N. Catsimpoolas (Plenum Press, New York) p. 141. Loos, H., B. Blok-Schut, R. Van Doorn, R. Hoksbergen, A.B. De la Rivi~re and L. Meerhof, 1976, Blood 48,731. Nathanson, S.D., P.L. Zamfirescu, S.I. Drwe and S. Wilbur, 1977, J. Immunol. Methods 18,225. Rinehart, J.J., B.J. Gormus, P. Lange and M.E. Kaplan, 1978, J. Immunol. Methods 23, 207. Sanderson, R.J., F.I. Shepperdson, A.E. Vatler and D.W. Talmage, 1977, J. Immunol. 118, 1409. Talstad, I. and J.S. Gundersen, 1979, Scand. J. Haematol. 23, 197. Ulmer, A.J. and H.D. Flad, 1979, J. Immunol. Methods 30, 1. Weiner, R.S. and V.O. Sha, 1980, J. Immunol. Methods 36, 89. Yam, L.T., C.Y. Li and W.H. Crosby, 1971, Am. J. Clin. Pathol. 55, 283.
31
MONOCYTE PURIFICATION WITH COUNTERFLOW C E N T R I F U G A T I O N M O N I T O R E D BY C O N T I N U O U S F L O W CYTOMETRY '
P.H.M. DE MULDER, J.M.C. WESSELS, D.A. ROSENBRAND, J.B.J.M. SMEULDERS, D.J.Th. WAGENER and C. HAANEN Division of Hematology, Department of Internal Medicine, University Hospital St. Radboud, Geert Grooteplein Zuid 16, 6525 GA Nijmegen, The Netherlands
(Received 29 March 1981, accepted 8 July 1981)
Continuous monitoring of cell light scatter during counterflow centrifugation of a mononuclear cell suspension allows counting and size recognition of the cell types elutriated. With this method an optimal separation point between monocytes and lymphocytes, determined for each individual donor, may be established. With a constant flow of 15 ml/min this separation point is found at centrifugal velocities ranging from 2348 to 2444 rpm (n = 10). From 50 ml venous blood, 84.1% + 4.1% (15.7 + 8.6 × 106 ) of all elutriated monocytes, with a purity of 92.4% + 1.4%, is collected in a volume of 50 + 1 ml. In the same run, 92% + 4.3% of the lymphocytes is gathered in one fraction with a purity of 98.9% + 0.7%. After counterflow centrifugation, 91.6 + 10.5% of the cells loaded is recovered; viability exceeds 98%. INTRODUCTION O p t i m a l s t u d y o f i m m u n o l o g i c a l and n o n - i m m u n o l o g i c a l f u n c t i o n s o f m o n o c y t e s requires purified m o n o c y t e suspensions. C o n v e n t i o n a l isolation t e c h n i q u e s are based u p o n either t h e i r a d h e r e n t p r o p e r t i e s (Koller et al., 1 9 7 3 ; A c k e r m a n and Douglas, 1 9 7 8 ; R i n e h a r t et al., 1 9 7 8 ) or d e n s i t y ( L o o s et al., 1 9 7 6 ; N a t h a n s o n et al., 1 9 7 7 ; U l m e r a n d Flad, 1 9 7 9 ) . M o n o c y t e isolat i o n b y c o u n t e r f l o w c e n t r i f u g a t i o n (elutriation) has r e c e n t l y been s h o w n t o give high p u r i t y , g o o d r e c o v e r y , and excellent viability a n d f u n c t i o n (Sanderson et al., 1 9 7 7 ; Weiner and Sha, 1 9 8 0 ; C o n t r e r a s et al., 1 9 8 0 ; L i o n e t t i et al., 1 9 8 0 ) . S e p a r a t i o n is based m a i n l y o n d i f f e r e n c e s in cell size a n d to a m u c h lesser e x t e n t o n d e n s i t y . M e t i c u l o u s r e g u l a t i o n o f flow, centrifugal f o r c e and t e m p e r a t u r e are essential t o achieve o p t i m a l p u r i f i c a t i o n . A f t e r relatively ' b l i n d ' e l u t r i a t i o n in f r a c t i o n s , screening o f t h e e f f l u e n t is usually p e r f o r m e d b y m e a n s o f a C o u l t e r c o u n t e r . In this p a p e r a n e w m e t h o d is described w h e r e b y c o n t i n u o u s m o n i t o r i n g o f t h e e l u t r i a t o r o u t p u t is o b t a i n e d , 1This study was supported by the Queen Wilhelmina Foundation, The Netherlands Organisation against Cancer (KWF/NOK). 0022-1759/81/0000--0000/$02.75 © 1981 Elsevier/North-Holland Biomedical Press
32 allowing simultaneous counting and size quantification. In this way it is possible to determine the optimal separation point between the different mononuclear cell populations in each elutriator procedure, resulting in improved efficiency of counterflow centrifugation. MATERIALS AND METHODS
Collection of blood Approximately 5 0 m l venous blood from healthy volunteers, anticoagulated with 6 IU/ml preservative-free heparin, is obtained b y venepuncture. An aliquot is taken for total and differential cell counting. Preparation of the mononuclear cell suspension (MNL ) The blood is immediately diluted 3-fold with calcium- and magnesium-free Hank's buffered balanced salt solution (HBBS), supplemented with 100 mg di-K-EDTA/1, final pH 7.3, 295 mosM/kg. Thirty-five ml samples are layered on 15 ml 1.077 g/ml Ficoll-Isopaque (pH 6.6, 272 mosM/kg). After centrifugation (400 X g; 30 min; 20°C) and removal of the supernatant, the interphase is collected, diluted with HBBS + 0.4% bovine serum albumin w/v (BSA), washed once ( 6 0 0 X g , 1 5 m i n , 4°C) and resuspended in 5 - - 8 m l HBBS + 0.4% BSA. A fixed volume is taken for counting and differentiation. The remaining cells are introduced into the elutriatior. Elu tria tion Elutriation system. This consists of a J2-21C centrifuge equipped with the JE-6 elutriator rotor and standard separation chamber (Beckman Instruments, Palo Alto, CA, U.S.A.). The flow is maintained with a roller pump (Masterflex4~ole-Parmer Instruments, Chicago, IL, U.S.A.) and pulsations are flattened b y a pulse suppressor chamber. Medium temperature is maintained at 10°C. The J2-21C centrifuge is modified by installing a fine scaled speed selector (one scale unit: 2.6 -+ 0.1 rpm) and replacing the C102 condenser (0.022 UF) b y a condenser of 47 pF; this results in improved stability of rotor speed. R p m measurement was altered by using a high resolution counter (120 HMZ pH 6667 Philips, Eindhoven, The Netherlands). The elutriator effluent is collected in a small d o w n f l o w cabinet. HBBS + 0.4% BSA w/v is used as elutriation medium. Cell recognition device (Fig. 1). Continuous sampling of the elutriator o u t p u t is established by means of a T-drain. This sample stream (a, 0.2 ml/ min) mantled in a sheath stream (b, 1.4 ml/min), is continuously analyzed for cell number and cell light scatter by the electro-optical peroxidase unit of the Hemalog-D (Technicon Instruments Corp., Tarrytown, U.S.A.). This unit, dismantled from the Hemalog-D, consists o f a flow cell with optical assembly for focusing the light from a tungsten halogen light source and an absorption and scatter photodiode. The light scatter signals are accumulated and displayed on an ND-100 multichannel analyzer (Nuclear Data Inc., Schaumberg,
33
1
2
3
Fig. 1. Schematic representation of elutriator rotor (1) and the cell recognition device (2), consisting of: (a) sample stream directly derived from elutriator effluent; (b)sheath flow; (c) roller p u m p ; (d) electro-optical unit; (e) ND-100 multichannel analyzer. Collection of elutriator o u t p u t (3).
IL, U.S.A.). The resulting histogram (Fig. 2) indicates the number o f cells as a function o f cell size (scatter). When a new cluster o f cells with a markedly different scatter signal is elutriated, a change in the histogram appears on the monitor. At any time the profile can be erased or stored in a m e m o r y , enabling direct comparison o f elutriated cell populations. Elutriation technique. Prior to introduction o f the cell suspension, entrapped air is removed, rotor speed adjusted to 2500 rpm, and medium flow established at 15 ml/min under control o f a flow-meter (Sho-rate flowmeter t y p e No. R-2-25-B Brooks Instruments Inc.). By means o f an infusor, the cell sample is introduced directly into the tubing line at a flow-rate of 0.3 ml/min. During this period m o n o c y t e s and larger l y m p h o c y t e s remain in the separation chamber, whereas smaller lymphocytes, erythrocytes and t h r o m b o c y t e s are eluted. When introduction o f the cells is completed the rotor speed is lowered 26 -+ 1 rpm every 2 min. The decrease o f centrifugal force results in an o u t f l o w o f l y m p h o c y t e s with increasing diameter, while the n u m b e r of cells eluted per time unit diminishes. At a certain rotor speed, indicated as the separation point, cells with a distinct scatter signal (monocytes) leave the elutriator (Fig. 2, profile 2). Since l y m p h o c y t e s are still being washed out, a double peaked histogram is seen, representing the t w o cell populations. The rotor speed is then immediately increased 15 rpm in order to wash o u t the remaining l y m p h o c y t e s and to retain the monocytes. Collection o f fraction 2 is started. When the number o f cells leaving the separation chamber falls below 100 cells/sec, the rotor is stopped and the effluent collected in one 50 ml t u b e (fraction 3). Immediately thereafter a 'rest' fraction is gathered (fraction 4). After washing with HBBS + 0.4% BSA (600 X g, 15 min, 4°C} the cells are resuspended and further analysis is performed. The whole procedure can be carried o u t within 1 h.
34
PRIOR
TO
ELUTRIATION
500
of
j, i
,
i
700
DURING
AFTER
500
0 0
II
500
| <
z
~o
a
6O0 CHANNEL SCATTER
60O
NUMBER (SIZE~
Fig. 2. Histograms as seen on the monitor of the ND-100 multiehannel analyzer. Prior to elutriation: the mononuclear cell suspension. During elutriation: profile 1 represents lymphocytes, profile 2 represents the mixed population and profile 3 monocytes. After elutriation: scatter analysis of the entire lymphocyte, mixed, and monocyte population.
35
Cell counting and differentiation Cell number in the starting material, MNL and elutriated fractions is measured with a Coulter counter. Cytocentrifuge preparations o f the MNL and elutriated fractions are made according to Talstad and Gundersen (1979) and stained with May-Griinwald-Giemsa and non-specific esterase, respectively (Yam et al., 1971; Lawrence and Grossman, 1979). Differential counting is performed on 400 cells. Cell viability Cell viability is assessed b y t w o methods. (1) Cells suspended in a concentration of 3--5 × 10S/ml are mixed with 20 #l fluorescein di-acetate/ml (5 mg FDA/ml acetone) and 20 pl ethidium bromide/ml (0.0025 mg EB/ NaC1 35.8 mg/ml and Tris 12.1 mg/ml, pH 7.5). Viable cells are FDA-positive (green cytoplasmic fluorescence; Celada and Rotman, 1967) and dead cells ethidium bromide-positive (red DNA fluorescence). (2) Exclusion o f trypan blue b y viable cells. RESULTS If the rotor speed is continuously decreased and the flow-rate maintained constant, cells with a gradually increasing diameter will leave the elutriator. With the effluent monitored by the cell recognition device measuring light scatter to analyse cell number and size, it is possible to record the distribution of these parameters in a mononuclear cell suspension (Fig. 3). In this particular experiment the monitoring was started after elutriation of the t h r o m b o c y t e s . The first and highest peak represents the lymphocytes, the second contains the monocytes. This form of cell scatter registration allows recognition of the different cells during elutriation. The possibility o f immediate identification of cell t y p e combined with low elutriator o u t p u t between given cell populations is the basis of this separation method. The histograms registered during elutriation are comparable with those of the final separated fractions (Fig. 2). The results of 10 runs are listed in Table 1. Fraction 3 consists of elutriated m o n o c y t e s of high purity with only slight admixture of l y m p h o c y t e s (6.5 + 1.6%), some basophils, and very few segmented neutrophils. The majority of the l y m p h o c y t e s is present in the first fraction with a purity of 98.9 -+ 0.7%, with only 1.1 + 0.6% monocytes. In the second fraction a mixed population is found: 2.6 + 4.3% of the lymphocytes and 6.2 -+ 2.8% of the elutriated monocytes. Finally a less pure m o n o c y t e fraction (80.8 -+ 5.6%) with 17.8 +- 4.5% l y m p h o c y t e s is gathered, representing only 5.5 -+ 3.1 and 0.5 + 0.3% respectively of the total eluted cell number. The rotor speed (rpm) at which, for the first time, two cell populations are seen, is defined as the separation point. This is found at 2392 -+ 39.5 rpm with a range of 2348--2444 rpm. Viability o f the elutriated cells is 97.7 + 1.1% as determined b y E B / F D A and 98.6 + 0.5% by trypan blue.
36
SCATTER
{SIZE)
Fig. 3. Relationship between cell number and light scatter signal of 44 consecutively elutriated fractions. Each of the 44 histograms represents the number and the corresponding light scatter of the cells elutriated in 150 sec. With the same interval the rotor speed was diminished 26 ± 1 rpm, while the flow remained constant at 15 ml/min. Fraction 1 is comparable to histograms 1--29; fraction 2 to histograms 29--32; fraction 3 and 4 to histograms 32--44. A f t e r e l u t r i a t i o n , 9 1 . 6 -+ 1 0 . 5 % o f t h e l o a d e d c e l l s a r e r e c o v e r e d . B y t h e m e t h o d d e s c r i b e d , 8 8 . 5 -+ 1 7 % o f t h e m o n o c y t e s a n d 6 4 . 8 + 9 % o f t h e l y m p h o c y t e s p r e s e n t in t h e p e r i p h e r a l b l o o d s a m p l e a r e r e c o v e r e d i n t h e e l u t r i a t o r e f f l u e n t , o f w h i c h 7 2 % a n d 6 0 % r e s p e c t i v e l y a r e f o u n d in t h e p u r e s t
TABLE 1 Purity and yield of lymphocytes and monocytes in the 4 elutriated fractions (mean % ± S.D.). Yield is expressed as the relative distribution of lymphocytes and monocytes over the 4 fractions; purity as the composition within each fraction. Fraction
1
2 3 4
Monocytes
Lymphocytes
Purity (%)
Yield (%)
Purity (%)
Yield (%)
4 . 1 ± 1.8 23.9±13.8 9 2 . 4 ± 1.4 8 0 . 8 ± 5.6
1.1±0.6 6.2±2.8 84.1±4.1 5.5±3.1
9 8 . 9 ± 0.7 75.4±13.7 6 . 5 ± 1.7 1 7 . 8 ± 4.5
92 ± 4 . 3 6 ±4.3 1.5±0.6 0.5±0.3
37 fractions. The absolute n u m b e r of m o n o c y t e s and l y m p h o c y t e s found in those fractions is (15.7 ± 8.6) X 106 and {63.9 ± 20) X 106 respectively. DISCUSSION Purified m o n o c y t e s are needed for study of their immunological and nonimmunological functions. Isolation on the basis of functional properties means selection and possible changes in cellular metabolism induced b y the isolation technique (Bodel et al., 1977). Counterflow centrifugation avoids this and provides an excellent m e t h o d for cell separation. Sanderson et al. (1977) were the first to report considerable purification o f a small quantity of m o n o c y t e s by elutriation. Weiner and Sha (1980), Lionetti et al. (1980), Contreras et al. (1980) and Figdor et al. (1981) described the isolation o f large quantities. A disadvantage o f the technique, however, is the relatively blind separation in fractions obtained at flow-rates and rotor speeds based upon results o f previous experiments. The use of a cell recognition device, continuously measuring elutriator o u t p u t , overcomes this problem. Firstly, one can actually see when a cohort of cells is collected. In our experiments fraction 1 is collected in 456 ± 36 ml, fraction 2 in 116 ± 56 ml and fractions 3 and 4 each in 50 -+ 1 ml. Secondly and even more important, the technique allows immediate recognition o f the eluted cell type. Contreras et al. (1980) stated that the elutriation characteristics of each donor's mononuclear cell suspension are different, and each run has to be tailored individually for each donor. The relatively wide range of rpm (2348--2444) found as the separation point in o u r study confirms this and corroborates the advantage of a separation m e t h o d involving direct visualization and recognition. On the other hand Fogelman et al. (1979) and Figdor et al. (1981) d o c u m e n t e d highly reproducible m o n o c y t e isolation from run to run w i t h o u t individual adjustment. In addition to the variability of flow, rotor speed and temperature already mentioned, which, if not standardized for each run, influence the elutriation characteristics considerably; biological spread in cell volume may account for the variation in separation point observed in the present study and m a y be of importance when dealing with patients' mononuclear cell suspensions. Using the isolation technique described, we achieved excellent purification o f m o n o c y t e s and l y m p h o c y t e s with good recovery and viability. Moreover the m o n o c y t e s are gathered in a small effluent volume (50 ml). The results reported here with a relatively small (50 ml) aliquot of peripheral venous blood as the source of the MNL suspensions and actual identification o f the eluted cell type, support the potential application of this purification technique in the study o f m o n o c y t e function in patients. Preliminary results on blood from patients with Hodgkin's disease, non-Hodgkin lymphomas, and infectious mononucleosis are essentially similar to those reported here.
38 ACKNOWLEDGEMENTS
We are indebted to A. Plas and L. Coenen for their skilful assistance with the installation and adjustement of the elutriation equipment and Technicon Instruments, Rotterdam, The Netherlands, for kindly supplying the peroxidase unit. We acknowledge J. Van Egmond's help with the computer operation. REFERENCES Ackerman, S.K. and S.D. Douglas, 1978, J. Immunol. 120, 1372. Bodel, P.T., B.A. Nichols and D.F. Baintaon, 1977, J. Exp. Med. 145,264. Celada, F. and B. Rotman, 1967, Proc. Nail. Acad. Sci. U.S.A. 57,630. Contreras, T.J., J.F. Jemionek, H.C. Stevenson, V.M. Hartwig and A.S. Fauci, 1980, Cell. Immunol. 54,215. Figdor, C.G., W.S. Bont, J.E. De Vries and W.L. Van Es, 1981, J. Immunol. Methods 40, 277. Fogelman, A.M., J. Seager, M. Hokom and P.A. Edwards, 1979, J. Lipid Res. 20,379. KoUer, C., G. Kind, P. Hurtibise, A. Sagone and A. Lobuglio, 1973, J. Immunol. 111, 1610. Lawrence, C. and R. Grossman, 1979, Stain Technol. 54,321. Lionetti, F.J., S.M. Hunt and C.R. Valeri, 1980, in: Methods of Cell Separation, Vol. 5, ed. N. Catsimpoolas (Plenum Press, New York) p. 141. Loos, H., B. Blok-Schut, R. Van Doorn, R. Hoksbergen, A.B. De la Rivi~re and L. Meerhof, 1976, Blood 48,731. Nathanson, S.D., P.L. Zamfirescu, S.I. Drwe and S. Wilbur, 1977, J. Immunol. Methods 18,225. Rinehart, J.J., B.J. Gormus, P. Lange and M.E. Kaplan, 1978, J. Immunol. Methods 23, 207. Sanderson, R.J., F.I. Shepperdson, A.E. Vatler and D.W. Talmage, 1977, J. Immunol. 118, 1409. Talstad, I. and J.S. Gundersen, 1979, Scand. J. Haematol. 23, 197. Ulmer, A.J. and H.D. Flad, 1979, J. Immunol. Methods 30, 1. Weiner, R.S. and V.O. Sha, 1980, J. Immunol. Methods 36, 89. Yam, L.T., C.Y. Li and W.H. Crosby, 1971, Am. J. Clin. Pathol. 55, 283.