Journal of Immunological Methods, 24 (1978) 389--393

389

© Elsevier/North-Holland Biomedical Press Short communication

A RAPID ONE-STEP PROCEDURE FOR PURIFICATION OF MONONUCLEAR AND POLYMORPHONUCLEAR LEUKOCYTES FROM HUMAN BLOOD USING A MODIFICATION OF THE HYPAQUE-FICOLL TECHNIQUE

A. FERRANTE and Y.H. THONG

Department of Paediatrics, The University of Adelaide, Adelaide Children's Hospital, Adelaide 5006, Australia (Received 10 September 1978, accepted 28 September 1978) A one-step procedure for purification of mononuclear and polymorphonuclear cells from human blood is described. It is a modification of the Hypaque-Ficoll method with density of 1.095 g/ml. Centrifugation at 200 x g for 20--30 min resulted in the separation of mononuclear and polymorphonuclear cells into 2 distinct bands at the interface. The mononuclear cell fraction contained 83.9 + 1.6% lymphocytes and 13.8 + 2.3% monocytes, while the other consisted of highly purified neutrophils (96.4 + 1.0%). Leukocyte recovery by this method was always greater than 80% and viability exceeded 98%. Both cell fractions retained their immunological functions.

INTRODUCTION The preparation of relatively pure populations of mononuclear(MN) and polymorphonuclear (PMN) leukocytes from blood is an important prerequisite for immunological studies in man. The method of B6yum (1968) involves the isolation of MN cells by Hypaque-Ficollcentrifugationas a first step, followed by dextran (Dewar, 1978) or gelatin (Anderson et al., 1978) sedimentation of red blood cells to obtain PMN. Others have used dextran sedimentation as a first step to obtain mixed leukocytes, and then separate MN from PMN by Hypaque-Ficoll centrifugation. A third step to lyse contaminating red blood cells with ammonium chloride is also required to obtain relativelypure PMN. We report here that by increasing the specific gravity of the HypaqueFicoll gradient from 1.080 g/ml (Bbyum, 1968) to 1.095 g/ml, the leukocytes separate as 2 bands on the interface while red blood cells lie at the bottom of the tube. Analysis of the leukocyte bands showed predominantly MN cells in the top band and PMN cells in the bottom band. MATERIALS AND METHODS Hypaque-Ficoll density gradient centrifugation An 85% solution of Hypaque (28.33% sodium 3,5-diacetamido-2,4,6-trii o d o b e n z o a t e ) was p u r c h a s e d f r o m W i n t h r o p L a b o r a t o r i e s , N e w S o u t h Wales,

390 Australia. Ficoll, a sucrose p o l y m e r (M.W. 4 0 0 , 0 0 0 , P h a r m a c i a , S w e d e n ) was dissolved in distilled w a t e r at a c o n c e n t r a t i o n of 9% w/v. T h e H y p a q u e Ficoll d e n s i t y g r a d i e n t was a m i x t u r e o f 20 ml H y p a q u e and 90 ml Ficoll s o l u t i o n . T h e m i x t u r e has a d e n s i t y of 1.095 g/ml. Sterile, disposable 10 ml plastic t u b e s were used for c e n t r i f u g a t i o n . T o each t e s t - t u b e was a d d e d 3 ml o f t h e H y p a q u e - F i c o l l gradient. T h e n 5--6 ml h e p a r i n i s e d h u m a n b l o o d o b t a i n e d b y v e n e p u n c t u r e was c a r e f u l l y layered on t o p o f the gradient, using a p i p e t t e . T h e t u b e was c e n t r i f u g e d at 200 x g for 2 0 - - 3 0 min at r o o m t e m p e r a t u r e . T h e 2 b a n d s at the interface were r e m o v e d s e p a r a t e l y with d i f f e r e n t p i p e t t e s and w a s h e d thrice with m e d i u m 199. Cell r e c o v e r y was d e t e r m i n e d b y c o u n t i n g in a h a e m o c y t o m e t e r b e f o r e a n d a f t e r c e n t r i f u g a t i o n . Cell p u r i t y was d e t e r m i n e d by staining on glass slides with M a y - G r u n w a l d / G i e m s a . Cell viability was d e t e r m i n e d b y exclusion o f t r y p a n blue.

Immunological function T h e f u n c t i o n a l integrity o f the MN b a n d was assayed b y m e a n s o f proliferative r e s p o n s e s to m i t o g e n s and allogeneic l y m p h o c y t e s ( T h o n g et al., 1973). T h e PMN b a n d was tested with regard to c h e m o t a x i s (Nelson et al., 1 9 7 5 ; T h o n g et al., 1978), p h a g o c y t o s i s ( T h o n g and F e r r a n t e , 1977), fungicidal c a p a c i t y ( T h o n g and F e r r a n t e , 1977), h e x o s e - m o n o p h o s p h a t e s h u n t activity ( T h o n g and Rencis, 1 9 7 7 ) and q u a n t i t a t i v e n e u t r o p h i l i o d i n a t i o n ( T h o n g a n d F e r r a n t e , 1978). RESULTS T w o d i s t i n c t b a n d s were o b s e r v e d at the i n t e r f a c e a f t e r 2 0 - - 3 0 min cent r i f u g a t i o n at 2 0 0 X g (Fig. 1). T h e t o p b a n d consisted o f m a i n l y MN cells, while the s e c o n d b a n d was p r e d o m i n a n t l y PMN l e u k o c y t e s (Table 1). T h e r e was no red b l o o d cell c o n t a m i n a t i o n ; these cells had m i g r a t e d to the b o t t o m o f the t u b e .

TABLE 1 CELL TYPES IN THE MONONUCLEAR AND POLYMORPHONUCLEAR CELL FRACTIONS Results are expressed as mean ± S.D. of 5 experiments using blood obtained from 5 individuals. Cell fractions Frl Fr2

Differential count (%) Lymphocytes

Monocytes

Basophils

Neutrophils

Eosinophils

83.9+1.6 1.2±0.4

13.8±2.3 --

0.5±0.5 -

1.820.8 96.4±1.0

-2.4±1.0

391

Fig. 1. Isolation of mononuclear (Fr 1) and polymorphonuclear ceils (Fr 2) by centrifugation of human blood on Hypaque-Ficoll (specific gravity 1.095) at 200 xg for 20--30 rain.

T o t a l l e u k o c y t e r e c o v e r y was greater than 80% in all e x p e r i m e n t s and cell viability e x c e e d e d 98%. T h e i m m u n o l o g i c a l f u n c t i o n o f l y m p h o c y t e s and n e u t r o p h i l s purified by this p r o c e d u r e r e m a i n e d i n t a c t (Tables 2 and 3).

392 'FABLE 2 I,YMPHOCYTE FUNCTION Results represent mean +_S.D. of 5 experiments. Assay

Mitogen phytohaemagglutinin Pokeweed mitogen Concanavalin A Mixed lymphocyte culture

[ 3H ]TdR incorporation Resting

Stimulated

1,794 1,794 1,794 1,223

47,595 _+ 13,086 29,680 _+ 6,687 42,450 +_13,496 10,346± 3,714

+ 1¢158 + 1,458 + 1,458 + 647

TABLE 3 NEUTROPHIL FUNCTION Neutrophil assay

Kinesis Chemotaxis a Random mobility b Phagocytosis c Fungicidal capacity d Iodinatione tlexose monophosphate shunt f

Results (mean + S.D. of 5 experiments)

1.89 0.70 74.5 95.1 5.4 9.5

+ 0.14 + 0.14 +_ 2.3 f. 1.2 +- 1.0 -+ 0.8

a,b Expressed as the migration distance (mm) of neutrophils towards the chemotactic well and control well in 3 h respectively. c Expressed as the % inhibition of [3H]uridine uptake (phagocytic index). d Expressed as the % of fungi killed by neutrophils after 5 h incubation. e Expressed as the quantity of iodination in 1 h in pmoles/10 7 neutrophils. f Expressed as the stimulation index and calculated as follows: cpm [1-14C]glucose in latex flask cpm [l-14C]glucose in resting flask

DISCUSSION T h e B S y u m m e t h o d ( 1 9 6 8 ) is a n e x c e l l e n t d e v i c e f o r p u r i f i c a t i o n o f m o n o n u c l e a r cells. T h e i s o l a t i o n o f g r a n u l o c y t e s , h o w e v e r , p r e s e n t s diffic u l t i e s b e c a u s e o f t h e high s p e c i f i c g r a v i t y o f t h e s e cells. S i m p l e s e d i m e n tation by d e x t r a n or m e t h y l c e l l u l o s e (Skoog and Beck, 1956) can provide g o o d y i e l d s o f l e u k o c y t e s , b u t p u r e M N a n d PMN p o p u l a t i o n s are n o t o b t a i n a b l e . T h e B S y u m m e t h o d ( 1 9 6 8 ) r e s u l t s in o n l y 50% cell r e c o v e r y . A r e c e n t m o d i f i c a t i o n (Dewar, 1 9 7 8 ) resulted in a higher yield {approxim a t e l y 70%) b u t t h e 3 - s t e p p r o c e d u r e is b o t h t e d i o u s a n d t i m e - c o n s u m i n g . T h e o n e - s t e p p r o c e d u r e d e s c r i b e d in t h i s p a p e r has s i g n i f i c a n t a d v a n t a g e s

393 o v e r o t h e r m e t h o d s . Firstly, it is s i m p l e and rapid, r e q u i r i n g o n l y 30 m i n f o r c o m p l e t i o n . S e c o n d l y , relatively p u r e p o p u l a t i o n s o f b o t h MN and PMN are p r o v i d e d b y a simple m a n o e u v r e . T h i r d l y , high yields o f b o t h cell t y p e s are o b t a i n e d . F o u r t h l y , t h e r e is n o red b l o o d cell c o n t a m i n a t i o n o f the granuloc y t e f r a c t i o n , so t h a t a m m o n i u m c h l o r i d e lysis a n d its possible adverse e f f e c t s on n e u t r o p h i l s is n o t r e q u i r e d . F i f t h l y , the i m m u n o l o g i c a l i n t e g r i t y o f the cells is preserved; it is possible t h a t a h e a l t h i e r cell p o p u l a t i o n results f r o m less h a n d l i n g and m o r e rapid s e p a r a t i o n . This m e t h o d has b e e n in use in o u r l a b o r a t o r y for m o r e t h a n 2 years a n d has b e e n e m p l o y e d in clinical as well as e x p e r i m e n t a l studies. REFERENCES Anderson, R., A. Glover and A.R. Robson, 1978, Immunology 35, 141. Btiyum, A., 1968, Scand. J. Clin. Lab. Invest. 21 (Suppl. 97), 77. Dewar, C., 1978, J. Immunol. Methods 20,301. Nelson, R.D., P.G. Quie and R.L. Simmons, 1975, J. Immunol. 115, 1650. Skoog, W.A. and W.S. Beck, 1956, Blood 11,436. Thong, Y.tl. and A. Ferrante, 1977, IRCS Med. Sci. 5,482. Thong, Y.H. and A. Ferrante, 1978, J. Immunol. Methods 20, 297. Thong, Y.H. and V. Rencis, 1977, Acta Paediat. Scand. 66, 757. Thong, Y.H., R.W. Steele, M.M. Vincent, S.A. ttensen and J.A. Bellanti, 1973, New Engl. J. Med. 289,604. Thong, Y.It., B.S. Douglas and A. Ferrante, 1978, Acta Paediat. Scand. 67,383.

A rapid one-step procedure for purification of mononuclear and polymorphonuclear leukocytes from human blood using a modification of the Hypaque-Ficoll technique.

Journal of Immunological Methods, 24 (1978) 389--393 389 © Elsevier/North-Holland Biomedical Press Short communication A RAPID ONE-STEP PROCEDURE F...
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