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Purification of human monocytes: Isolation and collection of large numbers of peripheral blood monocytes
Journal of Immunological Methods, 36 (1980) 89--97 © Elsevier/North-Holland Biomedical Press
89
PURIFICATION OF HUMAN MONOCYTES: ISOLATION AND COLLECTION OF LARGE NUMBERS OF PERIPHERAL BLOOD MONOCYTES i
ROY S. WEINER and VIRENDRA O. SHAH Department of Medicine, Division of Medical Oncology, University of Florida College of Medicine, Gainesville, FL 32610, U.S.A. (Received 4 February 1980, accepted 12 March 1980)
Monocytes from human peripheral blood were purified by elutriation centrifugation. Up to 1.5 x 109 peripheral blood mononuclear cells could be separated in isotonic media to yield 6--8 x 108 lymphocytes with 95% purity and 1.5--2.5 x 108 monocytes with greater than 90% purity. The temperature at which elutriation was performed determined the purity of the monocyte fraction. Both the lymphocyte and monocyte fractions were characterized by cell sizing and histochemical staining, Most of the myeloperoxidase positive mononuclear cells (monocytes) had a modal volume of 378 pm 3 while 10% were within the modal volume of the lymphocytes, 131 pm 3. Thus, there are two populations of cells with histochemical properties of monocytes which are separable by size. The collection and isolation of human peripheral blood monocyte populations in large numbers will facilitate studies of their functional characteristics.
INTRODUCTION The i m p o r t a n c e o f t h e m o n o c y t e - m a c r o p h a g e class o f cells is b e c o m i n g increasingly a p p a r e n t in m a n y areas o f cell-mediated i m m u n o l o g i c a l research, It is generally a c c e p t e d t h a t t h e ability to isolate a n d store h u m a n l y m p h o c y t e s facilitates t h e sequential testing o f i m m u n o l o g i c c o m p e t e n c e o f p a t i e n t s u n d e r g o i n g i m m u n o s u p p r e s s i v e or i m m u n o s t i m u l a t o r y t h e r a p e u t i c m a n i p u l a t i o n . Little is k n o w n t o date c o n c e r n i n g t h e value o f m o n o c y t e assays in m o n i t o r i n g h o s t responses in h u m a n p a t i e n t s with cancer a n d o t h e r diseases. Successful isolation and ability t o harvest large n u m b e r s o f peripheral b l o o d m o n o c y t e s b y t e c h n i q u e s w h i c h are well t o l e r a t e d b y n o r m a l d o n o r s a n d b y p a t i e n t s w o u l d facilitate basic studies o f their f u n c t i o n a n d i n t e r a c t i o n s with o t h e r cells. This p a p e r deals with such t e c h n i q u e s f o r isolation o f l y m p h o c y t e s a n d m o n o c y t e s . T h e r e are several m e t h o d s available f o r p u r i f y i n g peripheral b l o o d m o n o c y t e s a n d l y m p h o c y t e s based either o n their ability t o a d h e r e t o glass or a plastic surface, or o n their d e n s i t y . These m e t h o d s f o r enriching m o n o c y t e s 1 This work was supported by NCI Contract No. N01-CB-74131.
90 have varying degrees of contaminating lymphocytes and polymorphonuclear leukocytes. The most widely used m e t h o d of separation uses the ability of monocytes to adhere to glass or plastic surfaces. But this method has several disadvantages: difficulty in quantitation, considerable loss of cell number and viability when cells are removed by mechanical or chemical means (Broderson and Burns, 1973), and activation and alterations in m o n o c y t e metabolism by the process of adherence (David, 1975; Bodel et al., 1977; Van Ginkel et al., 1977). Several investigators have developed means to separate monocytes from other leukocytes in density gradients using either continuous or discontinuous gradients of albumin, Ficoll, sucrose, and/or silica polyvinylpyrrolidinone (Bennet and Cohn, 1966; Noble and Cutts, 1968; Brubaker and Evans, 1969; Loos et al., 1976; Barr et al., 1977; Nathanson et al., 1977). These methods, however, allow handling only of small quantities of cells or require several hours for completion. The present study reports the isolation and collection of monocytes in large quantities and high purity from normal human blood using only isotonic medium and with prior adherence. MATERIALS AND METHODS Normal donors were leukopheresed on the Haemonetic Model 30 semicontinuous cell separator as previously described (Weiner et al., 1977). Briefly, acid-citrate-dextrose (ACD) solution was added to the whole blood at the outflow tubing from the donor's arm. The filling rate of the bowl was 60--80 ml/min until the buffy coat reached the shoulder at the top of the bowl. The rate was then slowed to 30 ml/min and when the buffy coat was near the hub of the bowl, the cell collection was begun and continued for 90 sec, yielding about 45 ml of cells. This process of cell collection was repeated 2--3 times. Platelets were removed by diluting the sample with 1.5 vol of Ca 2-, Mg2- free Hanks' Balanced Salt Solution (HBSS), containing 5 units per ml preservative free heparin and centrifuging at 150 × g for 15 min. The top two-thirds of the supernatant containing 70% of the platelets was discarded after verifying that it was leukocyte poor. Mononuclear cells were purified from erythrocytes and granulocytes by isopyknic centrifugation over Ficoll-Hypaque (S.G. 1.078) by the m e t h o d of BSyum (1968). The mononuclear cells at the interface were removed and washed twice in HBSS. The final cell pellet was resuspended in 5 ml of HBSS for loading into the counter-flow centrifuge. From a small aliquot, cell count and cytocentrifuge smears were done. About 1--1.5 × 109 mononuclear cells were loaded into the elutriator chamber. An elutriator rotor equipped with a specially designed separation chamber (Sanderson et al., 1976) in a Beckman J-21C centrifuge was used for the isolation of lymphocytes and monocytes. The elution medium was HBSS, Ca 2÷- and Mg2÷-free, containing 100 mg EDTA/1. The elutriation rotor, chamber, and all tubing were washed with ethyl alcohol followed by sterile
91 water before each run. The flow rate was calibrated at the beginning of each run. A centrifuge speed of 2500 rpm (625 × g) was used. Experiments were carried o u t with the rotor at r o o m temperature (22--24°C) and at 10°C. The cells were eluted at a flow rate of 15--29 ml/min. Cells eluted from the centrifuge were collected in 400 ml vol and centrifuged at 300 × g for 10 min in 250 ml conical centrifuge tubes. The cells were washed one more time in HBSS. The cell pellet was resuspended in HBS8 with Ca 2÷ and Mg 2÷ containing 5% human AB serum and cells were counted and stained with May-Gruenwald Giemsa, non-specific esterase (NSE) (Knowles et al., 1978), and peroxidase (Kaplow, 1965). Three hundred cells were evaluated morphologically. Cell volume was measured in a Coulter Counter fitted with a channelizer. The electronic cell volume frequency distributions were obtained on unfixed cells with a Coulter Counter Model ZBI (70 pm diameter orifice) interfaced with a 100-channel pulse height analyzer and an X-Y plotter (General Electronics, Inc.). The system was calibrated using 10.12 pm polystyrene spheres. The modal volume, MV (the electronic cell volume corresponding to the peak channel), was calculated in each case. All frequency distribution curves were standardized on 4000 cells in the peak channel. RESULTS The pheresis of whole blood yielded 82% of the monocytes and 90% of the l y m p h o c y t e s in a 510 ml volume (Table 1). The b u f f y coats from 2--4 units of pheresed blood provided 1.5--2.5 × 109 mononuclear cells after isopyknic centrifugation to eliminate erythrocytes and granulocytes and a second, slow centrifugation to eliminate platelets. Using a constant rotor speed of 2500 rpm (625 X g), the cells were elutriated at flow rates between 15 and 29 ml/min in 2 ml/min increments. Preliminary data indicated that at a flow rate of 23 ml/min, more than 95% of the elutriated cells contained myeloperoxidase positive cells. The yield, however, was only approximately 40%. Experiments were performed to determine whether the peroxidase negative cells could be purged at a slow flow rate with minimum loss of monocytes thus permitting elutriation of monocytes at a higher flow rate in both high yield and high purity. With the rotor at room temperature at 17 ml/ rain, 59% of the total cells were elutriated. Less than 5% of the cells stained positive with myeloperoxidase. At 22 ml/min, 17% of the total cells were elutriated and this population was greater than 88% monocytes by the criteria of positive histochemical staining for myeloperoxidase and for nonspecific esterase. About 1% of the cells were recoverable at 28 ml/min and these represented a mixture of myeloperoxidase positive and myeloperoxidase negative cells. About 23% of the cells remained unaccounted for. Significant clumping was observed in the elutriation chamber at the end of the procedure. Experiments were then performed to determine whether
92 TABLE 1 Yield of cells from pheresis of whole blood X10 s . Pheresis performed using ACD as anticoagulant. Flow rate was 30 ml/min at the time of collection. Cell numbers are based on a calculated pheresed volume of 510 ml of peripheral blood. Numbers in ( ) are the % of cells in the pheresed volume.
Lymphocytes Monocytes Granulocytes
Pre
1st 30 ml
2nd 30 ml
7.7 2.8 11.3
6.26 (80%) 1.90 (68%) 1.07 (9%)
0.8 (10%) 0.4 (14%) 1.1 (10%)
s e p a r a t i o n c o u l d be carried o u t at a colder t e m p e r a t u r e . T h e r o t o r was c o o l e d to 1 0 ° C a n d e l u t r i a t i o n carried o u t o n c e again at 625 × g at f l o w r a t e s o f 17 m l / m i n , 22 m l / m i n , a n d 28 m l / m i n . A t 17 m l / m i n , 66% of t h e t o t a l cells w e r e e l u t r i a t e d . O n l y 5% o f t h e cells stained f o r m y e l o p e r o x i d a s e . A t 22 m l / m i n 18% o f t h e t o t a l cells w e r e e l u t r i a t e d a n d this p o p u l a t i o n was g r e a t e r t h a n 94% m o n o c y t e s b y the criteria o f positive h i s t o c h e m i c a l staining f o r m y e l o p e r o x i d a s e a n d n o n - s p e c i f i c esterase. Less t h a n 2% o f t h e cells were r e m o v e d at 28 m l / m i n a n d o n c e again, these w e r e a m i x t u r e o f p e r o x i d a s e positive a n d negative cells. O n l y 13% o f t h e cells r e m a i n e d u n a c c o u n t e d f o r (Table 2). T h e t e m p e r a t u r e at w h i c h t h e e l u t r i a t i o n was carried o u t sign i f i c a n t l y i n f l u e n c e d t h e p u r i t y o f m o n o c y t e s . T h e cells o b t a i n e d in Fract i o n 2 w e r e 94% m o n o c y t e s w h e n t h e r o t o r t e m p e r a t u r e was 10°C as c o m p a r e d to 88% m o n o c y t e s w i t h t h e r o t o r t e m p e r a t u r e at 2 2 - - 2 4 ° C. T h e yield o f l y m p h o c y t e s a n d m o n o c y t e s in t h e t w o cell-rich f r a c t i o n s is d i s p l a y e d in T a b l e 3. A t a r o t o r t e m p e r a t u r e o f 10°C, 85% o f t h e l y m p h o c y t e s w e r e r e c o v e r e d in F r a c t i o n 1 a n d 70% o f t h e m o n o c y t e s were recovere r e d in F r a c t i o n 2. T h e p e r c e n t a g e o f l y m p h o c y t e s r e c o v e r e d in F r a c t i o n 2 was significantly i n f l u e n c e d b y t h e r o t o r t e m p e r a t u r e . While 5.9% o f t h e t o t a l l y m p h o c y t e s w e r e p r e s e n t in F r a c t i o n 2 at 2 2 - - 2 4 ° C , o n l y 1.3% o f t h e t o t a l l y m p h o c y t e s w e r e p r e s e n t in F r a c t i o n 2 at 10°C. T h e t o t a l m o n o c y t e p o p u l a t i o n was p a r t i t i o n e d u n e q u a l l y a m o n g t h e t w o cell-rich f r a c t i o n s . S e v e n t y p e r c e n t o f t h e m o n o c y t e s w e r e r e c o v e r e d in F r a c t i o n 2 while 12% w e r e r e c o v e r e d in F r a c t i o n 1 being e l u t r i a t e d w i t h t h e b u l k o f t h e l y m p h o c y t e s . T h e viability o f t h e l y m p h o c y t e s a n d m o n o c y t e s regularly e x c e e d e d 95% as d e t e r m i n e d b y t r y p a n blue e x c l u s i o n . T h e m o d a l v o l u m e o f t h e cells l o a d e d i n t o the c h a m b e r is s h o w n in Fig. 1A. T h e large p e a k at 131 # m 3 c o n t a i n s m o s t l y l y m p h o c y t e s . T h e m o n o c y t e s w h i c h are a b o u t 25% o f t h e p o p u l a t i o n are n o t discernible. A f t e r e l u t r i a t i o n , h o w e v e r , it is clear t h a t t h e cells r e c o v e r e d at 17 m l / m i n (Fig. 1B) have t h e s a m e m o d a l v o l u m e as t h e F i c o l l - H y p a q u e s a m p l e , w h e r e a s t h e cells r e c o v e r e d at 22 m l / m i n have a m o d a l v o l u m e of 378 p m 3 (Fig. 1C). T h e 22 m l / m i n f r a c t i o n is m u c h m o r e h e t e r o g e n e o u s w i t h
93 TABLE2 E l u t r i a t i o n o f m o n o n u c l e a r cells ( m e a n ± S.E.M.). M o n o n u c l e a r cells w e r e o b t a i n e d b y pheresis o f peripheral b l o o d and were cleared o f platelets b y c e n t r i f u g a t i o n and o f e r y t h r o c y t e s and g r a n u l o c y t e s by i s o p y k n i c c e n t r i f u g a t i o n over F i c o l l - H y p a q u e (S.G. : 1.078). The centrifugal f o r c e o f t h e r o t o r was 625 x g (2500 r p m ) , and the e l u t r i a t i o n v o l u m e 400 ml for each f r a c t i o n . F l o w rate (ml/min) L o a d e d cells F r a c t i o n no. 1
17
F r a c t i o n no. 2
22
F r a c t i o n no. 3
28
T o t a l cells X 10 8
Recovery 1 (%)
Purity 2 (%)
10.8 11.7 6.5 7.8 1.7 2.1 0.1 0.2
100 100 59.2 66.3 16.6 18.2 1.2 2.0
22.8 25.0 4.9 4.5 87.8 94.0 N.T. N.T.
+ 0.6 a -+ 1.0 b ± 0.6 a -+ 0.8 b ± 0.1 a ± 0.3 b + 0.05 a ± 0.10 b
± 3.0 -+ 3.0 ± 1.0 -+ 2.0 ± 0.5 -+ 1.1
± 0.7 ± 0.7 ± 0.9 -+ 0.9 -+ 1.6 c + 1.0 c d
1 R e c o v e r y was t h e % o f t o t a l cells l o a d e d i n t o the c h a m b e r . 2 P u r i t y was t h e % o f m o n o c y t e s as d e t e r m i n e d by staining for m y e l o p e r o x i d a s e and nonspecific esterase. a R o t o r t e m p e r a t u r e was 2 2 ° - - 2 4 ° C (18 e x p e r i m e n t s ) . b R o t o r t e m p e r a t u r e was 10°C (7 e x p e r i m e n t s ) . c p < 0.05 b y analysis o f covariance using the % o f m o n o c y t e s in t h e l o a d e d cell populat i o n as a covariate. d Not tested.
respect to volume than the 17 ml/min fraction. The cell diameters as calculated from the cell volumes indicate that the lymphocytes are 6.31 pm and the
major
monocyte
peak
is
at
8.97
gm.
The
exclusion
diameter
for
TABLE 3 Yield o f l y m p h o c y t e s and m o n o c y t e s o b t a i n e d b y elutriation (%). Centrifugal f o r c e was 625 x g ( 2 5 0 0 r p m ) ; e l u t r i a t i o n volume was 400 ml for each fraction.
Lymphocytes Monocytes
Fraction 2 b
73.7 85.2 12.8 12.2
5.9 1.3 63.5 69.5
_+ 4.1 ± 3.4 ± 2.3 ± 2.4
c d c d
+ 1.0 e -4- 0.2 e ± 4.6 ± 6.6
1 : cells r e c o v e r e d a f t e r e l u t r i a t i o n at 17 m l / m i n . F r a c t i o n 2: cells r e c o v e r e d after e l u t r i a t i o n at 22 m l / m i n . R o t o r at r o o m t e m p e r a t u r e (18 e x p e r i m e n t s ) . R o t o r at 10°C (7 e x p e r i m e n t s ) . p = 0.003 b y M a n n W h i t n e y U test.
a Fraction
b c d e
Fraction 1 a
94
I00
/
75
50
25
V
I00
I/
B
75 to
H b
50
z 25
0 --
K
, __
i
i
°°
z5
0
~
ss
,,o
,~5
2~0
2~5
33o
385
44o
49s
VOLUME ~ 3
Fig, 1. V o l u m e s p e c t r a o b t a i n e d f r o m a C o u l t e r c o u n t e r a n d e h a n n e l i z e r for m o n o n u c l e a r cell p o p u l a t i o n s . A: m o n o n u c l e a r cell p o p u l a t i o n l o a d e d i n t o t h e e l u t r i a t i o n c h a m b e r . B: m o n o n u e l e a r c e l l p o p u l a t i o n e l u t r i a t e d at 1 7 m l / m i n f l o w rate ( s p e e d = 2 5 0 0 r p m ) . C : m o n o n u e l e a r cell p o p u l a t i o n e l u t r i a t e d at 2 2 m | ] m i n f l o w rate ( s p e e d = 2 5 0 0 r p m ) .
95 17 ml/min is less than 8.2 pm, whereas the exclusion diameter at 22 ml/min is less than 9.1 pm using the 2500 rpm centrifuge speed. DISCUSSION The present study reports the feasibility of collecting large numbers of h u m a n mononuclear cells and using elutriation centrifugation to separate l y m p h o c y t e s from monocytes. Using a chamber developed by Sanderson et al. (1976) in the elutriation rotor, 1--1.5 × 109 Ficoll-Hypaque separated cells can be processed in less than 2 h. While more cells are readily obtainable by pheresis, the chamber capacity limits each run to fewer than 2 × 109 cells. Unacceptable cell clumping results when higher concentrations are loaded into the chamber. Each elutriation run, however, can provide more than 2 × 108 monocytes contaminated by about 5% lymphocytes. It is feasible to perform two or even three runs in a laboratory day. The entire procedure is performed in isotonic media. Adherence of the monocytes is minimized by using Ca 2÷- and Mg2÷-free HBSS and adding EDTA. Our data expand upon those of Sanderson et al. (1977) and differ significantly in two respects. Firstly, we eluted the cells at a pre-determined flow rate w i t h o u t monitoring the cell size. Our data indicates that m o n o c y t e isolation is highly reproducible from run to run. We eluted the cells in a sufficiently large volume so that recovery was predictable and uniform at each flow rate. Secondly, modal volume and flow rates of our elutriated l y m p h o c y t e - and monocyte-rich fractions were significantly different from those reported by Sanderson et al. (1977). We f o u n d the monocyte-rich fraction to have a modal volume of 378 pm 3 and Sanderson reported 360 pm 3. These volumes are well within tolerable differences considering the heterogeneity of the monocyte-rich population. However, Sanderson eluted the monocyte-rich fraction at 28 ml/min and found that a 20 ml/min flow rate resulted in a m o n o c y t e to l y m p h o c y t e ratio approaching 1 : 2 (Sanderson et al., 1977). We found very few cells elutable at 28 ml/min and our purest m o n o c y t e fraction was recoverable at the 22 ml/min flow rate. Several other authors have reported values of 150--198 pm 3 as the modal volume of normal lymphocytes (Ben-Sasson et al., 1974; Braylan et al., 1978). Our lymphocyte-rich fraction had a modal volume of 131 pm 3 rather than 320 pm 3 as reported by Sanderson et al. (1977). This discrepancy has been resolved. Sanderson's initial data was based on a Coulter Counter calibration error. In fact, the modal volume of lymphocytes was 180 pm 3 and the modal volume of the monocyte-rich fraction was 420 pm 3 (Sanderson, personal communication). Recently, Fogelman et al. (1979) has obtained purified monocytes using the elutriation technique as well. The purest fractions reported by these authors were obtained under conditions similar to those used by us. However, we were able to recover 87% of the mononuclear cells in the two cellular fractions, while Fogelman et al. (1979) had a 50% loss of cells by the
96 m e t h o d t h e y report. This discrepancy may be due to the small volume of their elutriation aliquots. Our early experiments with elutriation volumes of 100 ml per aliquot resulted in increased c o n t a m i n a t i o n of our m o n o c y t e fraction with l y m p h o c y t e s . We f o u n d that prolonging the 17 ml/min flow rate and using larger volumes of elutriation media increased the recovery of l y m p h o c y t e s t h e r e b y decreasing the n u m b e r of l y m p h o c y t e s available to be eluted with the m o n o c y t e s at 22 ml/min. Four hundred milliliters was f o u n d to be adequate. Fogelman et al. (1979) report elutriation aliquots of only 40 ml which m ay be inadequate to clear all the cells elutable at a given flow rate. The effect o f r o t o r t e m p e r a t u r e on the elutriation procedure is significant. Compared with 22--24°C, a r o t o r t e m p e r a t u r e of 10°C eliminates cell clumping within the r o t o r , decreases the recovery of l y m p h o c y t e s in the m o n o c y t e - r i c h fraction, and t h e r e b y increases the purity of m o n o c y t e s recovered at a flow rate of 22 ml/min. There is no evidence t hat the cooler t e m p e r a t u r e affects the elutriation properties of the m o n o c y t e s . Rather, there is a tr en d towards increasing the recovery of the l y m p h o c y t e s in the fraction eluted at 17 ml/min. The h e t e r o g e n e i t y of cells with respect to size t hat are retained in the c h amb er between 17 and 22 ml/min flow rates contrast to the h o m o g e n e i t y o f their myeloperoxidase positivity. It should be possible to recover populations o f cells between 18 and 21 ml/min and study their physical and functional properties. There is a significant population of myeloperoxidase positive cells which contaminates the l y m p h o c y t e s and which are less than 8.2 pm in diameter or less than 288 pm 3 in volume. The functional characteristics of this m o n o c y t e population requires definition. A variety o f gradient centrifugation techniques have been used to isolate m o n o c y t e s , but by and large their yields and purities are inferior to the elutriation m e t h o d described above. We have combined cytopheresis and elutriation centrifugation to obtain morphologically intact normal human m o n o c y t e s , in suspension, with a high puri t y and in large quantities. Cells can be readily obtained to s t udy hum an m o n o c y t e funct i on in normal subjects and in patients with a variety of disease states. REFERENCES Barr, R.P., T. Whang-Peng and S. Perry, 1977, Biomedicine 26,112. Ben-Sasson, S., D. Patinkin, W.B. Grover and F. Koljanski, 1974, J. Cell. Physiol. 84,205. Bennet, W.E. and Z.A. Cohn, 1966, J. Exp. Med. 123,145. Bodel, P.T., B.A. Nichols and D.F. Baintaon, 1977, J. Exp. Med. 145,264. B6yum, A., 1968, Scand. J. Clin. Invest. 21 (Suppl. 97), 1. Braylan, R.C., B.J. Fowlkes, E.S. Jaffe, S.K. Sanders, C.W. Berard and C.J. Herman, 1978, Cancer 41,201. Broderson, M.P. and C.P. Burns, 1973, Proc. Soc. Exp. Biol. Med. 144, 941. Brubaker, L.H. and W.H. Evans, 1969, J. Lab. Clin. Med. 73, 1036. David, J.R., 1975, Fed. Proc. 34, 1730.
97 Fogelman, A.M, J. Seager, M. Hokum and P.A. Edwards, 1979, J. Lipid Res. 20, 379. Kaplow, L.S., 1965, Blood 26,215. Knowles, D.M., T. Hoffman, M. Ferrarini and H.G. Kunkel, 1978, Cell. Immunol. 35, 112. Loos, H., B. Blok-Schut, R. Van Doorn, R. Hoksbergen, A.B. de la Riviere and L. Meerhaf, 1976, Blood 48,731. Nathanson, S.D., P.L. Zamfrescu, S.I. Drew and S. Wilbur, 1977, J. Immunol. Methods 18,225. Noble, P.B. and J.H. Cutts, 1968, J. Lab. Clin. Med. 72, 533. Sanderson, R.J., K.E. Bird, N.F. Palmer and J. Brenman, 1976, Anal. Biochem. 71,615. Sanderson, R.J., F.T. Shepperdson, A.E. Vatter and D.W. Talmage, 1977, J. Immunol. 118, 1409. Van Ginkel, C.J.W., W.G. Van Aken, J.I.H. Oh and J. Vreeken, 1977, Br. J. Haematol. 37, 35. Weiner, R.S., C.M. Richman and R.A. Yankee, 1977, Blood 49,391.
89
PURIFICATION OF HUMAN MONOCYTES: ISOLATION AND COLLECTION OF LARGE NUMBERS OF PERIPHERAL BLOOD MONOCYTES i
ROY S. WEINER and VIRENDRA O. SHAH Department of Medicine, Division of Medical Oncology, University of Florida College of Medicine, Gainesville, FL 32610, U.S.A. (Received 4 February 1980, accepted 12 March 1980)
Monocytes from human peripheral blood were purified by elutriation centrifugation. Up to 1.5 x 109 peripheral blood mononuclear cells could be separated in isotonic media to yield 6--8 x 108 lymphocytes with 95% purity and 1.5--2.5 x 108 monocytes with greater than 90% purity. The temperature at which elutriation was performed determined the purity of the monocyte fraction. Both the lymphocyte and monocyte fractions were characterized by cell sizing and histochemical staining, Most of the myeloperoxidase positive mononuclear cells (monocytes) had a modal volume of 378 pm 3 while 10% were within the modal volume of the lymphocytes, 131 pm 3. Thus, there are two populations of cells with histochemical properties of monocytes which are separable by size. The collection and isolation of human peripheral blood monocyte populations in large numbers will facilitate studies of their functional characteristics.
INTRODUCTION The i m p o r t a n c e o f t h e m o n o c y t e - m a c r o p h a g e class o f cells is b e c o m i n g increasingly a p p a r e n t in m a n y areas o f cell-mediated i m m u n o l o g i c a l research, It is generally a c c e p t e d t h a t t h e ability to isolate a n d store h u m a n l y m p h o c y t e s facilitates t h e sequential testing o f i m m u n o l o g i c c o m p e t e n c e o f p a t i e n t s u n d e r g o i n g i m m u n o s u p p r e s s i v e or i m m u n o s t i m u l a t o r y t h e r a p e u t i c m a n i p u l a t i o n . Little is k n o w n t o date c o n c e r n i n g t h e value o f m o n o c y t e assays in m o n i t o r i n g h o s t responses in h u m a n p a t i e n t s with cancer a n d o t h e r diseases. Successful isolation and ability t o harvest large n u m b e r s o f peripheral b l o o d m o n o c y t e s b y t e c h n i q u e s w h i c h are well t o l e r a t e d b y n o r m a l d o n o r s a n d b y p a t i e n t s w o u l d facilitate basic studies o f their f u n c t i o n a n d i n t e r a c t i o n s with o t h e r cells. This p a p e r deals with such t e c h n i q u e s f o r isolation o f l y m p h o c y t e s a n d m o n o c y t e s . T h e r e are several m e t h o d s available f o r p u r i f y i n g peripheral b l o o d m o n o c y t e s a n d l y m p h o c y t e s based either o n their ability t o a d h e r e t o glass or a plastic surface, or o n their d e n s i t y . These m e t h o d s f o r enriching m o n o c y t e s 1 This work was supported by NCI Contract No. N01-CB-74131.
90 have varying degrees of contaminating lymphocytes and polymorphonuclear leukocytes. The most widely used m e t h o d of separation uses the ability of monocytes to adhere to glass or plastic surfaces. But this method has several disadvantages: difficulty in quantitation, considerable loss of cell number and viability when cells are removed by mechanical or chemical means (Broderson and Burns, 1973), and activation and alterations in m o n o c y t e metabolism by the process of adherence (David, 1975; Bodel et al., 1977; Van Ginkel et al., 1977). Several investigators have developed means to separate monocytes from other leukocytes in density gradients using either continuous or discontinuous gradients of albumin, Ficoll, sucrose, and/or silica polyvinylpyrrolidinone (Bennet and Cohn, 1966; Noble and Cutts, 1968; Brubaker and Evans, 1969; Loos et al., 1976; Barr et al., 1977; Nathanson et al., 1977). These methods, however, allow handling only of small quantities of cells or require several hours for completion. The present study reports the isolation and collection of monocytes in large quantities and high purity from normal human blood using only isotonic medium and with prior adherence. MATERIALS AND METHODS Normal donors were leukopheresed on the Haemonetic Model 30 semicontinuous cell separator as previously described (Weiner et al., 1977). Briefly, acid-citrate-dextrose (ACD) solution was added to the whole blood at the outflow tubing from the donor's arm. The filling rate of the bowl was 60--80 ml/min until the buffy coat reached the shoulder at the top of the bowl. The rate was then slowed to 30 ml/min and when the buffy coat was near the hub of the bowl, the cell collection was begun and continued for 90 sec, yielding about 45 ml of cells. This process of cell collection was repeated 2--3 times. Platelets were removed by diluting the sample with 1.5 vol of Ca 2-, Mg2- free Hanks' Balanced Salt Solution (HBSS), containing 5 units per ml preservative free heparin and centrifuging at 150 × g for 15 min. The top two-thirds of the supernatant containing 70% of the platelets was discarded after verifying that it was leukocyte poor. Mononuclear cells were purified from erythrocytes and granulocytes by isopyknic centrifugation over Ficoll-Hypaque (S.G. 1.078) by the m e t h o d of BSyum (1968). The mononuclear cells at the interface were removed and washed twice in HBSS. The final cell pellet was resuspended in 5 ml of HBSS for loading into the counter-flow centrifuge. From a small aliquot, cell count and cytocentrifuge smears were done. About 1--1.5 × 109 mononuclear cells were loaded into the elutriator chamber. An elutriator rotor equipped with a specially designed separation chamber (Sanderson et al., 1976) in a Beckman J-21C centrifuge was used for the isolation of lymphocytes and monocytes. The elution medium was HBSS, Ca 2÷- and Mg2÷-free, containing 100 mg EDTA/1. The elutriation rotor, chamber, and all tubing were washed with ethyl alcohol followed by sterile
91 water before each run. The flow rate was calibrated at the beginning of each run. A centrifuge speed of 2500 rpm (625 × g) was used. Experiments were carried o u t with the rotor at r o o m temperature (22--24°C) and at 10°C. The cells were eluted at a flow rate of 15--29 ml/min. Cells eluted from the centrifuge were collected in 400 ml vol and centrifuged at 300 × g for 10 min in 250 ml conical centrifuge tubes. The cells were washed one more time in HBSS. The cell pellet was resuspended in HBS8 with Ca 2÷ and Mg 2÷ containing 5% human AB serum and cells were counted and stained with May-Gruenwald Giemsa, non-specific esterase (NSE) (Knowles et al., 1978), and peroxidase (Kaplow, 1965). Three hundred cells were evaluated morphologically. Cell volume was measured in a Coulter Counter fitted with a channelizer. The electronic cell volume frequency distributions were obtained on unfixed cells with a Coulter Counter Model ZBI (70 pm diameter orifice) interfaced with a 100-channel pulse height analyzer and an X-Y plotter (General Electronics, Inc.). The system was calibrated using 10.12 pm polystyrene spheres. The modal volume, MV (the electronic cell volume corresponding to the peak channel), was calculated in each case. All frequency distribution curves were standardized on 4000 cells in the peak channel. RESULTS The pheresis of whole blood yielded 82% of the monocytes and 90% of the l y m p h o c y t e s in a 510 ml volume (Table 1). The b u f f y coats from 2--4 units of pheresed blood provided 1.5--2.5 × 109 mononuclear cells after isopyknic centrifugation to eliminate erythrocytes and granulocytes and a second, slow centrifugation to eliminate platelets. Using a constant rotor speed of 2500 rpm (625 X g), the cells were elutriated at flow rates between 15 and 29 ml/min in 2 ml/min increments. Preliminary data indicated that at a flow rate of 23 ml/min, more than 95% of the elutriated cells contained myeloperoxidase positive cells. The yield, however, was only approximately 40%. Experiments were performed to determine whether the peroxidase negative cells could be purged at a slow flow rate with minimum loss of monocytes thus permitting elutriation of monocytes at a higher flow rate in both high yield and high purity. With the rotor at room temperature at 17 ml/ rain, 59% of the total cells were elutriated. Less than 5% of the cells stained positive with myeloperoxidase. At 22 ml/min, 17% of the total cells were elutriated and this population was greater than 88% monocytes by the criteria of positive histochemical staining for myeloperoxidase and for nonspecific esterase. About 1% of the cells were recoverable at 28 ml/min and these represented a mixture of myeloperoxidase positive and myeloperoxidase negative cells. About 23% of the cells remained unaccounted for. Significant clumping was observed in the elutriation chamber at the end of the procedure. Experiments were then performed to determine whether
92 TABLE 1 Yield of cells from pheresis of whole blood X10 s . Pheresis performed using ACD as anticoagulant. Flow rate was 30 ml/min at the time of collection. Cell numbers are based on a calculated pheresed volume of 510 ml of peripheral blood. Numbers in ( ) are the % of cells in the pheresed volume.
Lymphocytes Monocytes Granulocytes
Pre
1st 30 ml
2nd 30 ml
7.7 2.8 11.3
6.26 (80%) 1.90 (68%) 1.07 (9%)
0.8 (10%) 0.4 (14%) 1.1 (10%)
s e p a r a t i o n c o u l d be carried o u t at a colder t e m p e r a t u r e . T h e r o t o r was c o o l e d to 1 0 ° C a n d e l u t r i a t i o n carried o u t o n c e again at 625 × g at f l o w r a t e s o f 17 m l / m i n , 22 m l / m i n , a n d 28 m l / m i n . A t 17 m l / m i n , 66% of t h e t o t a l cells w e r e e l u t r i a t e d . O n l y 5% o f t h e cells stained f o r m y e l o p e r o x i d a s e . A t 22 m l / m i n 18% o f t h e t o t a l cells w e r e e l u t r i a t e d a n d this p o p u l a t i o n was g r e a t e r t h a n 94% m o n o c y t e s b y the criteria o f positive h i s t o c h e m i c a l staining f o r m y e l o p e r o x i d a s e a n d n o n - s p e c i f i c esterase. Less t h a n 2% o f t h e cells were r e m o v e d at 28 m l / m i n a n d o n c e again, these w e r e a m i x t u r e o f p e r o x i d a s e positive a n d negative cells. O n l y 13% o f t h e cells r e m a i n e d u n a c c o u n t e d f o r (Table 2). T h e t e m p e r a t u r e at w h i c h t h e e l u t r i a t i o n was carried o u t sign i f i c a n t l y i n f l u e n c e d t h e p u r i t y o f m o n o c y t e s . T h e cells o b t a i n e d in Fract i o n 2 w e r e 94% m o n o c y t e s w h e n t h e r o t o r t e m p e r a t u r e was 10°C as c o m p a r e d to 88% m o n o c y t e s w i t h t h e r o t o r t e m p e r a t u r e at 2 2 - - 2 4 ° C. T h e yield o f l y m p h o c y t e s a n d m o n o c y t e s in t h e t w o cell-rich f r a c t i o n s is d i s p l a y e d in T a b l e 3. A t a r o t o r t e m p e r a t u r e o f 10°C, 85% o f t h e l y m p h o c y t e s w e r e r e c o v e r e d in F r a c t i o n 1 a n d 70% o f t h e m o n o c y t e s were recovere r e d in F r a c t i o n 2. T h e p e r c e n t a g e o f l y m p h o c y t e s r e c o v e r e d in F r a c t i o n 2 was significantly i n f l u e n c e d b y t h e r o t o r t e m p e r a t u r e . While 5.9% o f t h e t o t a l l y m p h o c y t e s w e r e p r e s e n t in F r a c t i o n 2 at 2 2 - - 2 4 ° C , o n l y 1.3% o f t h e t o t a l l y m p h o c y t e s w e r e p r e s e n t in F r a c t i o n 2 at 10°C. T h e t o t a l m o n o c y t e p o p u l a t i o n was p a r t i t i o n e d u n e q u a l l y a m o n g t h e t w o cell-rich f r a c t i o n s . S e v e n t y p e r c e n t o f t h e m o n o c y t e s w e r e r e c o v e r e d in F r a c t i o n 2 while 12% w e r e r e c o v e r e d in F r a c t i o n 1 being e l u t r i a t e d w i t h t h e b u l k o f t h e l y m p h o c y t e s . T h e viability o f t h e l y m p h o c y t e s a n d m o n o c y t e s regularly e x c e e d e d 95% as d e t e r m i n e d b y t r y p a n blue e x c l u s i o n . T h e m o d a l v o l u m e o f t h e cells l o a d e d i n t o the c h a m b e r is s h o w n in Fig. 1A. T h e large p e a k at 131 # m 3 c o n t a i n s m o s t l y l y m p h o c y t e s . T h e m o n o c y t e s w h i c h are a b o u t 25% o f t h e p o p u l a t i o n are n o t discernible. A f t e r e l u t r i a t i o n , h o w e v e r , it is clear t h a t t h e cells r e c o v e r e d at 17 m l / m i n (Fig. 1B) have t h e s a m e m o d a l v o l u m e as t h e F i c o l l - H y p a q u e s a m p l e , w h e r e a s t h e cells r e c o v e r e d at 22 m l / m i n have a m o d a l v o l u m e of 378 p m 3 (Fig. 1C). T h e 22 m l / m i n f r a c t i o n is m u c h m o r e h e t e r o g e n e o u s w i t h
93 TABLE2 E l u t r i a t i o n o f m o n o n u c l e a r cells ( m e a n ± S.E.M.). M o n o n u c l e a r cells w e r e o b t a i n e d b y pheresis o f peripheral b l o o d and were cleared o f platelets b y c e n t r i f u g a t i o n and o f e r y t h r o c y t e s and g r a n u l o c y t e s by i s o p y k n i c c e n t r i f u g a t i o n over F i c o l l - H y p a q u e (S.G. : 1.078). The centrifugal f o r c e o f t h e r o t o r was 625 x g (2500 r p m ) , and the e l u t r i a t i o n v o l u m e 400 ml for each f r a c t i o n . F l o w rate (ml/min) L o a d e d cells F r a c t i o n no. 1
17
F r a c t i o n no. 2
22
F r a c t i o n no. 3
28
T o t a l cells X 10 8
Recovery 1 (%)
Purity 2 (%)
10.8 11.7 6.5 7.8 1.7 2.1 0.1 0.2
100 100 59.2 66.3 16.6 18.2 1.2 2.0
22.8 25.0 4.9 4.5 87.8 94.0 N.T. N.T.
+ 0.6 a -+ 1.0 b ± 0.6 a -+ 0.8 b ± 0.1 a ± 0.3 b + 0.05 a ± 0.10 b
± 3.0 -+ 3.0 ± 1.0 -+ 2.0 ± 0.5 -+ 1.1
± 0.7 ± 0.7 ± 0.9 -+ 0.9 -+ 1.6 c + 1.0 c d
1 R e c o v e r y was t h e % o f t o t a l cells l o a d e d i n t o the c h a m b e r . 2 P u r i t y was t h e % o f m o n o c y t e s as d e t e r m i n e d by staining for m y e l o p e r o x i d a s e and nonspecific esterase. a R o t o r t e m p e r a t u r e was 2 2 ° - - 2 4 ° C (18 e x p e r i m e n t s ) . b R o t o r t e m p e r a t u r e was 10°C (7 e x p e r i m e n t s ) . c p < 0.05 b y analysis o f covariance using the % o f m o n o c y t e s in t h e l o a d e d cell populat i o n as a covariate. d Not tested.
respect to volume than the 17 ml/min fraction. The cell diameters as calculated from the cell volumes indicate that the lymphocytes are 6.31 pm and the
major
monocyte
peak
is
at
8.97
gm.
The
exclusion
diameter
for
TABLE 3 Yield o f l y m p h o c y t e s and m o n o c y t e s o b t a i n e d b y elutriation (%). Centrifugal f o r c e was 625 x g ( 2 5 0 0 r p m ) ; e l u t r i a t i o n volume was 400 ml for each fraction.
Lymphocytes Monocytes
Fraction 2 b
73.7 85.2 12.8 12.2
5.9 1.3 63.5 69.5
_+ 4.1 ± 3.4 ± 2.3 ± 2.4
c d c d
+ 1.0 e -4- 0.2 e ± 4.6 ± 6.6
1 : cells r e c o v e r e d a f t e r e l u t r i a t i o n at 17 m l / m i n . F r a c t i o n 2: cells r e c o v e r e d after e l u t r i a t i o n at 22 m l / m i n . R o t o r at r o o m t e m p e r a t u r e (18 e x p e r i m e n t s ) . R o t o r at 10°C (7 e x p e r i m e n t s ) . p = 0.003 b y M a n n W h i t n e y U test.
a Fraction
b c d e
Fraction 1 a
94
I00
/
75
50
25
V
I00
I/
B
75 to
H b
50
z 25
0 --
K
, __
i
i
°°
z5
0
~
ss
,,o
,~5
2~0
2~5
33o
385
44o
49s
VOLUME ~ 3
Fig, 1. V o l u m e s p e c t r a o b t a i n e d f r o m a C o u l t e r c o u n t e r a n d e h a n n e l i z e r for m o n o n u c l e a r cell p o p u l a t i o n s . A: m o n o n u c l e a r cell p o p u l a t i o n l o a d e d i n t o t h e e l u t r i a t i o n c h a m b e r . B: m o n o n u e l e a r c e l l p o p u l a t i o n e l u t r i a t e d at 1 7 m l / m i n f l o w rate ( s p e e d = 2 5 0 0 r p m ) . C : m o n o n u e l e a r cell p o p u l a t i o n e l u t r i a t e d at 2 2 m | ] m i n f l o w rate ( s p e e d = 2 5 0 0 r p m ) .
95 17 ml/min is less than 8.2 pm, whereas the exclusion diameter at 22 ml/min is less than 9.1 pm using the 2500 rpm centrifuge speed. DISCUSSION The present study reports the feasibility of collecting large numbers of h u m a n mononuclear cells and using elutriation centrifugation to separate l y m p h o c y t e s from monocytes. Using a chamber developed by Sanderson et al. (1976) in the elutriation rotor, 1--1.5 × 109 Ficoll-Hypaque separated cells can be processed in less than 2 h. While more cells are readily obtainable by pheresis, the chamber capacity limits each run to fewer than 2 × 109 cells. Unacceptable cell clumping results when higher concentrations are loaded into the chamber. Each elutriation run, however, can provide more than 2 × 108 monocytes contaminated by about 5% lymphocytes. It is feasible to perform two or even three runs in a laboratory day. The entire procedure is performed in isotonic media. Adherence of the monocytes is minimized by using Ca 2÷- and Mg2÷-free HBSS and adding EDTA. Our data expand upon those of Sanderson et al. (1977) and differ significantly in two respects. Firstly, we eluted the cells at a pre-determined flow rate w i t h o u t monitoring the cell size. Our data indicates that m o n o c y t e isolation is highly reproducible from run to run. We eluted the cells in a sufficiently large volume so that recovery was predictable and uniform at each flow rate. Secondly, modal volume and flow rates of our elutriated l y m p h o c y t e - and monocyte-rich fractions were significantly different from those reported by Sanderson et al. (1977). We f o u n d the monocyte-rich fraction to have a modal volume of 378 pm 3 and Sanderson reported 360 pm 3. These volumes are well within tolerable differences considering the heterogeneity of the monocyte-rich population. However, Sanderson eluted the monocyte-rich fraction at 28 ml/min and found that a 20 ml/min flow rate resulted in a m o n o c y t e to l y m p h o c y t e ratio approaching 1 : 2 (Sanderson et al., 1977). We found very few cells elutable at 28 ml/min and our purest m o n o c y t e fraction was recoverable at the 22 ml/min flow rate. Several other authors have reported values of 150--198 pm 3 as the modal volume of normal lymphocytes (Ben-Sasson et al., 1974; Braylan et al., 1978). Our lymphocyte-rich fraction had a modal volume of 131 pm 3 rather than 320 pm 3 as reported by Sanderson et al. (1977). This discrepancy has been resolved. Sanderson's initial data was based on a Coulter Counter calibration error. In fact, the modal volume of lymphocytes was 180 pm 3 and the modal volume of the monocyte-rich fraction was 420 pm 3 (Sanderson, personal communication). Recently, Fogelman et al. (1979) has obtained purified monocytes using the elutriation technique as well. The purest fractions reported by these authors were obtained under conditions similar to those used by us. However, we were able to recover 87% of the mononuclear cells in the two cellular fractions, while Fogelman et al. (1979) had a 50% loss of cells by the
96 m e t h o d t h e y report. This discrepancy may be due to the small volume of their elutriation aliquots. Our early experiments with elutriation volumes of 100 ml per aliquot resulted in increased c o n t a m i n a t i o n of our m o n o c y t e fraction with l y m p h o c y t e s . We f o u n d that prolonging the 17 ml/min flow rate and using larger volumes of elutriation media increased the recovery of l y m p h o c y t e s t h e r e b y decreasing the n u m b e r of l y m p h o c y t e s available to be eluted with the m o n o c y t e s at 22 ml/min. Four hundred milliliters was f o u n d to be adequate. Fogelman et al. (1979) report elutriation aliquots of only 40 ml which m ay be inadequate to clear all the cells elutable at a given flow rate. The effect o f r o t o r t e m p e r a t u r e on the elutriation procedure is significant. Compared with 22--24°C, a r o t o r t e m p e r a t u r e of 10°C eliminates cell clumping within the r o t o r , decreases the recovery of l y m p h o c y t e s in the m o n o c y t e - r i c h fraction, and t h e r e b y increases the purity of m o n o c y t e s recovered at a flow rate of 22 ml/min. There is no evidence t hat the cooler t e m p e r a t u r e affects the elutriation properties of the m o n o c y t e s . Rather, there is a tr en d towards increasing the recovery of the l y m p h o c y t e s in the fraction eluted at 17 ml/min. The h e t e r o g e n e i t y of cells with respect to size t hat are retained in the c h amb er between 17 and 22 ml/min flow rates contrast to the h o m o g e n e i t y o f their myeloperoxidase positivity. It should be possible to recover populations o f cells between 18 and 21 ml/min and study their physical and functional properties. There is a significant population of myeloperoxidase positive cells which contaminates the l y m p h o c y t e s and which are less than 8.2 pm in diameter or less than 288 pm 3 in volume. The functional characteristics of this m o n o c y t e population requires definition. A variety o f gradient centrifugation techniques have been used to isolate m o n o c y t e s , but by and large their yields and purities are inferior to the elutriation m e t h o d described above. We have combined cytopheresis and elutriation centrifugation to obtain morphologically intact normal human m o n o c y t e s , in suspension, with a high puri t y and in large quantities. Cells can be readily obtained to s t udy hum an m o n o c y t e funct i on in normal subjects and in patients with a variety of disease states. REFERENCES Barr, R.P., T. Whang-Peng and S. Perry, 1977, Biomedicine 26,112. Ben-Sasson, S., D. Patinkin, W.B. Grover and F. Koljanski, 1974, J. Cell. Physiol. 84,205. Bennet, W.E. and Z.A. Cohn, 1966, J. Exp. Med. 123,145. Bodel, P.T., B.A. Nichols and D.F. Baintaon, 1977, J. Exp. Med. 145,264. B6yum, A., 1968, Scand. J. Clin. Invest. 21 (Suppl. 97), 1. Braylan, R.C., B.J. Fowlkes, E.S. Jaffe, S.K. Sanders, C.W. Berard and C.J. Herman, 1978, Cancer 41,201. Broderson, M.P. and C.P. Burns, 1973, Proc. Soc. Exp. Biol. Med. 144, 941. Brubaker, L.H. and W.H. Evans, 1969, J. Lab. Clin. Med. 73, 1036. David, J.R., 1975, Fed. Proc. 34, 1730.
97 Fogelman, A.M, J. Seager, M. Hokum and P.A. Edwards, 1979, J. Lipid Res. 20, 379. Kaplow, L.S., 1965, Blood 26,215. Knowles, D.M., T. Hoffman, M. Ferrarini and H.G. Kunkel, 1978, Cell. Immunol. 35, 112. Loos, H., B. Blok-Schut, R. Van Doorn, R. Hoksbergen, A.B. de la Riviere and L. Meerhaf, 1976, Blood 48,731. Nathanson, S.D., P.L. Zamfrescu, S.I. Drew and S. Wilbur, 1977, J. Immunol. Methods 18,225. Noble, P.B. and J.H. Cutts, 1968, J. Lab. Clin. Med. 72, 533. Sanderson, R.J., K.E. Bird, N.F. Palmer and J. Brenman, 1976, Anal. Biochem. 71,615. Sanderson, R.J., F.T. Shepperdson, A.E. Vatter and D.W. Talmage, 1977, J. Immunol. 118, 1409. Van Ginkel, C.J.W., W.G. Van Aken, J.I.H. Oh and J. Vreeken, 1977, Br. J. Haematol. 37, 35. Weiner, R.S., C.M. Richman and R.A. Yankee, 1977, Blood 49,391.