Evaluation of rosette formation on smears

Journal of Immunological Methods, 14 (1977) 371--380 © Elsevier/North-Holland Biomedical Press

EVALUATION

OF ROSETTE

FORMATION

371

ON SMEARS

G. STEIN i

Department of Clinical Physiology, University of Ulm, Germany

A method of rosette evaluation on smears is described based on fixation of rosettes in suspension with glutaraldehyde after the incubation period. By this procedure many cell suspensions can be analysed under identical conditions at the same time. There is no need for immediate reading, and excellent permanent documentation is possible. In a relatively short time a thousand cells can be scored and the results recorded as histograms of attached erythrocytes instead of as empirically defined percentages.

INTRODUCTION S h e e p e r y t h r o c y t e s , u n t r e a t e d ( J o n d a l e t al., 1 9 7 2 ; W y b r a n e t al., 1 9 7 2 ) o r t r e a t e d w i t h n e u r a m i n i d a s e ( W e i n e r e t al., 1 9 7 3 ) o r t h e s u l f h y d r y l r e a g e n t A E T * ( K a p l a n a n d C l a r k , 1 9 7 4 ) a r e u s e d as a m a r k e r f o r h u m a n T c e l l s . R e d blood cells of various species coated with IgM-antibody and complement are u s e d t o d e t e c t c e l l s w i t h r e c e p t o r s f o r c o m p l e m e n t ( B i a n c o e t al., 1 9 7 0 ; R o s s e t al., 1 9 7 3 ) . E r y t h r o c y t e s p r e t r e a t e d w i t h I g G a n t i b o d y a r e u s e d t o d e t e c t Fc r e c e p t o r s ( F r O l a n d e t a l . , 1 9 7 4 ) . R e s u l t s a r e u s u a l l y e v a l u a t e d f r o m w e t p r e p a r a t i o n s a n d a c e r t a i n n u m b e r o f e r y t h r o c y t e s b o u n d t o l y m p h o c y t e s is t a k e n t o d e f i n e a r o s e t t e f o r m i n g c e l l , o f t e n as a l y m p h o c y t e b i n d i n g a t l e a s t 3 indicator erythrocytes. Wet preparations require immediate scoring after incubation and when more than one or two cell suspensions are being e x a m i n e d i t is a s s u m e d t h a t t h e n u m b e r o f r o s e t t e s d o e s n o t c h a n g e o n p r o longation of the incubation time. To keep this time relatively constant the n u m b e r o f cells e x a m i n e d h a s t o b e q u i t e l o w . I n a d d i t i o n , i t is a r b i t r a r y t o base assessment of positivity or negativity on attachment of a threshold n u m b e r of r e d cells. More informative are histograms of numbers of attached erythrocytes. But t o c o n s t r u c t t h e s e is d i f f i c u l t a n d l a b o r i o u s w i t h w e t p r e p a r a t i o n s . M o s t a t t e m p t s t o e v a l u a t e r o s e t t e f o r m a t i o n o n s m e a r s h a v e so f a r b e e n u n s a t i s f a c t o r y b e c a u s e o f d i s r u p t i o n o f t h e r o s e t t e s d u r i n g p r e p a r a t i o n . We f o u n d t h a t this problem can be solved by fixation of the lymphocyte--erythrocyte mixt u r e in s u s p e n s i o n a n d d e s c r i b e in t h i s p a p e r a s i m p l e a n d r e l i a b l e m e t h o d o f I Present address: II. Universit~its-Frauenklinik, Lindwurmstrasse 2a, 8 Mfinchen 2, Germany. * AET = S-(2-aminoethyl)-isothiuroniumbromide.

372 evaluating r o s e t t e f o r m a t i o n on smears, applicable to b o t h stable and fragile rosettes and allowing investigation o f m a n y cell suspensions at the same time. MATERIALS AND METttODS

Prepa ration of ly mp t7ocy les H e p a r i n i z e d v e n o u s b l o o d o f h e a l t h y adult b l o o d d o n o r s and o f a child with severe c o m b i n e d i m m u n o d e f i c i e n c y b e f o r e and a f t e r b o n e m a r r o w t r a n s p l a n t a t i o n ( G o l d m a n n et al., 1976) was first m i x e d with 0 . 0 0 5 g per ml o f c a r b o n y l iron p o w d e r (Faguet, 1974) and i n c u b a t e d at 38°C for 40 rain with c o n t i n u o u s shaking. A f t e r dilution 1 : 4 with tissue culture m e d i u m the b l o o d was l a y e r e d on F i c o l l - - I s o p a q u e solution and spun for 40 min at 4 0 0 g a c c o r d i n g to the m e t h o d o f BCyum (1968). ttalf o f the t o p layer was r e m o v e d and stored f r o z e n for use as diluted a u t o l o g o u s plasma (see below). T h e rest was r e m o v e d t o g e t h e r with the cells o f the interphase layer, diluted with tissue c u l t u r e m e d i u m b u f f e r e d with Hepes and Tris (MEM) and spun f o r 10 min at 250 g. T h e cells in the pellet were washed twice by resuspension with MEM s u p p l e m e n t e d with 5% h e a t inactivated calf serum (MEM 5% CS) and c e n t r i f u g a t i o n as above. Finally the cell c o n c e n t r a t i o n was adjusted to 2.5 × 106 cells per ml. F o r H E A - r o s e t t e f o r m a t i o n (see below) the l y m p h o c y t e s were finally r e s u s p e n d e d in MEM w i t h o u t serum. The m o r p h o l ogy o f the cells was c o n t r o l l e d on c y t o c e n t r i f u g e smears stained with M a y - G r t i n w a l d - - G i e m s a and with c y t o c h e m i c a l d e m o n s t r a t i o n o f a c~-naphthylesterase and p e r o x i d a s e to i d e n t i f y m o n o c y t i c cells (Yam, 1974).

Preparation of indicator erythrocytes C o m m e r c i a l l y available sheep b l o o d (Behringwerke) was used e i t h e r fresh as delivered or a f t e r up to 4 weeks storage at 4°C. The sheep b l o o d was diluted with MEM c o n t a i n i n g heparin and the SRBC were washed 4 times by c e n t r i f u g a t i o n for 10 min at 250 g and r e s u s p e n d e d in MEM. F o r E-rosettes the SRBC were r e s u s p e n d e d in MEM c o n t a i n i n g 5% foetal calf serum (CS). F o r A E T - r o s e t t e s SRBC were i n c u b a t e d in t h e A E T reagent for 15 rain at 37°C and a f t e r 4 washings r e s u s p e n d e d in MEM 5% CS. T h e reagent was prepared by dissolving 0.5 g A E T (Serva, Heidelberg) in 12.5 ml distilled water, the pH being adjusted t o 9.0 with 4 N NaOH. F o r EAC-rosettes d e t e c t i n g r e c e p t o r s for c o m p l e m e n t , SRBC were first i n c u b a t e d in MEM with trypsin (Serva, Heidelberg) for 60 rain at 37°C, and a f t e r 3 washings i n c u b a t e d in a subagglutinating dilution o f a n t i - S R B C - h e m o l y s i n (Behring A m b o c e p t o r 1 : 4 0 0 0 ) for 60 min 37°C. A f t e r 3 f u r t h e r washings the sensitized cells were i n c u b a t e d in fresh m o u s e serum (1 : 10 dilution) for 30 rain at 37°C and a f t e r 3 washes finally r e s u s p e n d e d in MEM 5% CS. All i n c u b a t i o n s were perf o r m e d at a c o n c e n t r a t i o n o f 10 ~ SRBC per ml. T h e final cell c o n c e n t r a t i o n s

373 w e r e a d j u s t e d to 125 × 106 S R B C / m l . F o r d e t e c t i o n o f r e c e p t o r s f o r h u m a n 7S i m m u n o g l o b u l i n (F~) h u m a n e r y t h r o c y t e s of b l o o d g r o u p O, R h positive, w e r e w a s h e d 5 t i m e s and i n c u b a t e d f o r 60 m i n at 37°C in a h u m a n s e r u m ( 1 : 4 d i l u t i o n ) c o n t a i n i n g anti-CD o f R i p l e y t y p e ( k i n d l y s u p p l i e d b y H. Stein, P a t h o l o g i c a l I n s t i t u t e , U n i v e r s i t y o f Kiel, G e r m a n y ) . A f t e r 3 washes these i n d i c a t o r cells w e r e r e s u s p e n d e d in MEM w i t h o u t s e r u m , the cell conc e n t r a t i o n was a d j u s t e d t o 50 × 106 p e r ml, a n d w e r e used f o r E A r o s e t t e s ( H E A r o s e t t e s ) as d e s c r i b e d b e l o w .

Rosette formalion F o r r o s e t t e f o r m a t i o n 0.1 ml o f t h e l y m p h o c y t e s u s p e n s i o n w e r e m i x e d with 0.1 ml o f o n e o f t h e e r y t h r o c y t e s u s p e n s i o n s in small r o u n d b o t t o m e d plastic t u b e s ( N U N C , R o s k i l d e , D e n m a r k ) . T h e i n c u b a t i o n c o n d i t i o n s were 1) E and A E T - r o s e t t e s : i m m e d i a t e c e n t r i f u g a t i o n 5 min at 150 g and incubation at r o o m t e m p e r a t u r e or at 4°C f o r 1 h, 2 h or o v e r n i g h t ) . H E A - r o s e t t e s : i m m e d i a t e c e n t r i f u g a t i o n a n d i n c u b a t i o n 30 min at r o o m t e m p e r a t u r e . 3) E A C - r o s e t t e s : i n c u b a t i o n 15 min at 3 7 ° C with c o n t i n u o u s shaking, f o l l o w e d b y c e n t r i f u g a t i o n a n d a d d i t i o n a l i n c u b a t i o n f o r 15 min at 37°C.

Termination o f incubation and evaluation o f results After the appropriate incubation period the lymphocyte~erythrocyte m i x t u r e s were r e s u s p e n d e d b y g e n t l y t a p p i n g t h e t u b e s a n d 0.8 ml o f a 0.8% g l u t a r a l d e h y d e s o l u t i o n in MEM was a d d e d giving a final c o n c e n t r a t i o n o f a b o u t 0.6% g l u t a r a l d e h y d e . A f t e r 20 m i n at t h e i n c u b a t i o n t e m p e r a t u r e 2 ml MEM were a d d e d and c y t o c e n t r i f u g e smears were m a d e f r o m o n e d r o p o f CS m i x e d with 0.2 ml o f t h e cell m i x t u r e s . T h e smears w e r e c h e c k e d b y m i c r o s c o p y f o r thin d i s t r i b u t i o n w i t h o u t c l u m p i n g at the edge, w h i c h was f o u n d to be a critical c o n d i t i o n . T h e s m e a r s w e r e stained in h e m a l u m f o r 5 rain, rinsed in t a p w a t e r for 10 min a n d m o u n t e d with e u k i t t . F o r each cell susp e n s i o n 500 cells were s c o r e d on 2 s m e a r s a n d t h e p e r c e n t a g e s of m o n o n u c l e a r cells with 0 - - 8 or m o r e a t t a c h e d e r y t h r o c y t e s were d e t e r m i n e d . G r a n u l o c y t e s were easily i d e n t i f i e d , b u t clear c u t d i f f e r e n t i a t i o n 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 was i m p o s s i b l e w i t h o u t a d d i t i o n a l m a n i p u l a t i o n such as p r i o r i n c u b a t i o n with latex particles. T h e results are r e c o r d e d e i t h e r as h i s t o g r a m s , or as p e r c e n t a g e o f R F C d e f i n e d as cells with 3 or m o r e and 5 or m o r e a t t a c h e d red cells. F o r c o m p a r i s o n with t h e c o n v e n t i o n a l e v a l u a t i o n o f R F C , w e t p r e p a r a t i o n s were s t u d i e d b y p u t t i n g a d r o p o f cell m i x t u r e on a slide, c o v e r i n g with a c o v e r slip a n d scoring 1 0 0 cells, using a p h a s e c o n t r a s t microscope.

Cell separation F o r s e p a r a t i o n into A E T - - R F C and R F C 5 ml o f a l y m p h o c y t e s u s p e n s i o n c o n t a i n i n g 5 × 106 cells/ml, were m i x e d w i t h 5 ml o f a A E T - - S R B C suspen-

374

sion (250 × 106 ml) in 25 ml siliconized glass tubes. After centrifugation for 10 rain at 150 g and incubation overnight at room temperature the mixture was gently resuspended, layered on Ficoll--Isopaque solution in 15 ml siliconized glass tubes and spun for 30 min at 400 g. The cells at the interphase layer and at the b o t t o m were collected, diluted with MEM and after centrifugation, resuspended in 1 : 4 diluted autologous heparinized plasma (see above). On incubation at 37°C complete lysis of the AET--SRBC occurred within 15 min owing to the extreme sensitivity of AET treated red cells to lysis by active complement. The cells were washed 3 times and prepared for rosette tests as described above. As control, a sample of unseparated cells was subjected to the same incubations, gradient centrifugation, and washing steps but incubated in MEM only instead mixing with AET--SRBC. RESULTS The cells isolated by incubation with carbonyl iron powder and Ficoll gradient centrifugation were 96--97% lymphocytes, 2% basophilic granulocytes, 0.5--1% neutrophils and 0.5--1% monocytes. The cell yield was about 60% of the blood lymphocytes. The recovery of cells after separation into AET--RFC and non-RFC was about 10% in the interphase layer and about 60% in the b o t t o m layer. Fig. 1

~L

m

f P

4

@ ,

~6

F i g . 1. C y t o c e n t r i f u g e

smear of AET-rosettes.

Q

375 100-

untreated

SRBC

80, m 60f. 9J

m --4

40-

o. 20

0 100-

1

2

3

4

AET t r e a t e d

5

6

7 =*8

6

7 •8

SRBC

80'

m

_e

o

60'

e

40-

20

0 "~ m

1

2

3

erythrocytes

4

S

attached

to

lymphocytea

Fig. 2. Histograms of rosette formation of blood lymphocytes with untreated and AETtreated SRBC after 1 h incubation at 4°C.

s h o w s t h e a p p e a r a n c e o f A E T - r o s e t t e s on smears p r e p a r e d b y t h e p r o c e d u r e described. In fig. 2 results o f r o s e t t e f o r m a t i o n w i t h u n t r e a t e d S R B C and A E T t r e a t e d S R B C are c o m p a r e d . T h e h i s t o g r a m s d e m o n s t r a t e t h a t w i t h A E T r o s e t t e f o r m a t i o n t h e r e is a clear d i s s o c i a t i o n o f cells with l o w a n d high n u m b e r s o f a t t a c h e d red cells. We f o u n d t h a t , as d e s c r i b e d b y o t h e r investigators, for optimal E-rosette formation with untreated SRBC prolonged i n c u b a t i o n at 4°C is n e c e s s a r y , b u t t h a t r o s e t t e f o r m a t i o n w i t h A E T t r e a t e d S R B C is equally g o o d at r o o m t e m p e r a t u r e as at 4°C a n d t h a t t h e r e is little d i f f e r e n c e b e t w e e n t h e e f f e c t s o f 1 h a n d 18 h i n c u b a t i o n . In a d d i t i o n , w h e r e a s E - r o s e t t e f o r m a t i o n decreases w h e n S R B C o l d e r t h a n 5--8 d a y s are used, A E T - r o s e t t e f o r m a t i o n is equally g o o d w i t h 4 w e e k s old S R B C as w i t h fresh cells, a n d t h e r e is n o d i f f e r e n c e in r o s e t t e f o r m a t i o n w h e t h e r t h e A E T - -

376

'°°lj AET

EAC

HEA

8O

6O

~oo]

0

2

4

6 ~8

0

2

4

6 i~8

0

2

4

6 IR8

0

2

4

6

1~8

0

2

4

6

k8

0

2

4

6

0

2

4

6 m8

0

2

4

6 a8

0

2

4

6 aB8

0

2

4

6 aR8

0

2

4

6 m8

0

2

4

6 18

2

4

6 ~8

0

2

4

6 R8

0

2

4

6 ="8

6O 4O 2

~'~

18

8O 60

e

a. lOO]

2

'1 0

erythrocytes attached to lymphocytes Fig. 3. Histograms o f the r o s e t t e f o r m a t i o n o f bh)od l y m p h o c y t e s with A E T - t r e a t e d SRBC (AET), SRBC c o a t e d with [gM-antih()dy and ('onlph,m('nL (EAC) ~)nd h u m a n O R h p~)s. e r y t h r o c y t e s c o a t e d with anti-CD (tIEA). Fiv0 norm~d blood donors.

S R B C are used i m m e d i a t e l y a f t e r A E T - t r e a t m e n t or a f t e r 5 d a y s storage at 4°C. T h u s , for d e t e c t i o n o f T cell r o s e t t e s we o b t a i n c o m m e r c i a l l y available s h e e p b l o o d o n c e a m o n t h a n d p r e p a r e A E T - - S R B C o n c e a week. In fig. 3 a n d t a b l e 1 t h e results o f r o s e t t e f o r m a t i o n in 15 e x p e r i m e n t s with l y m p h o c y t e s o f h e a l t h y b l o o d d o n o r s are given to d e m o n s t r a t e the range o f n o r m a l values o b t a i n e d . Fig. 4 gives the results in a p a t i e n t with severe c o m b i n e d i m m u n o d e f i c i e n c y b e f o r e and a f t e r b o n e m a r r o w t r a n s p l a n t a t i o n . This case and the i n t e r p r e t a t i o n a c c o r d i n g to the disease is d e s c r i b e d in detail elsewhere (Goldm a n n et al., 1976). It e x e m p l i f i e s t h e p a t h o l o g i c a l findings with the described r o s e t t e e v a l u a t i o n t e c h n i q u e . Fig. 5 c o m p a r e s r o s e t t e f o r m a t i o n a f t e r g r a d i e n t s e p a r a t i o n into a A E T - R F C rich and A E T - - R F C d e p r i v e d (:ell suspension. T h e h i s t o g r a m s d e m o n -

377 TABLE 1 Percent rosette forming cells and Ig positive cells in healthy blood donors. Lymphocytes isolated by incubation with carbonyl iron powder and ficoll gradient centrifugation. Exp.

AET-RFC

EAC-RFC

33E

35E

33E

1 2 3 4 5

80.4 86.0 91.4 86.4 87.2

74.6 80.6 84.0 83.6 81.2

. . . 28.0 23.0

6

HEA-RFC 35E

.

Ig pos.

33E

.

.

16.3 16.8

. . . ---

. .

. .

35E .

. . . ---

18 --

79.8

76.4

13.8

10.4

--

--

--

7 8 9 10 :l 1 12 13 14 15

74.8 68.8 88.6 75.8 77.8 81.4 85.8 87.6 74.8

62.4 60.5 86.0 70.0 73.8 78.0 82.4 85.6 64.8

25.4 21.2 . . 26.6 23.0 25.2 34.0 35.6

11.4 10.0

---

---

--:-

n

15

15

10

10

m -+ SD

81.8 6.4

76.3 8.4

25.6 6.7

17.3 7.5

. .

. . 23.8 12.0 13.2 28.0 31.0

. . 32.0 31.6 13.2 30.6 28.4

.

. 26.6 28.6 10.2 29.6 28.0

5

5

3

27.2 7.9

24.6 8.1

20.3

--15 28 --

-- = not tested.

strate the good separation resulting from the strong rosette formation with A E T - - R F C . M o s t cells o f t h e b o t t o m l a y e r f o r m s t r o n g r o s e t t e s w i t h m o r e t h a n 8 e r y t h r o c y t e s . In c o n t r a s t o n l y o c c a s i o n a l l y are s u c h s t r o n g A E T r o s e t t e s f o u n d a m o n g t h e i n t e r f a c e l a y e r cells. M o s t o f t h e l a t t e r ar e w i t h o u t r e d cells o r h a v e o n l y t w o o r t h r e e a d h e r e n t r e d cells. T h u s , in A E T t e s t s b i n d i n g o f 3 - - 5 r e d cells s e e m s t o b e n o n s p e c i f i c a n d m u s t b e c o u n t e d as n e g a t i v e . E A C t e s t e d r o s e t t e f o r m a t i o n w i t h m o r e t h a n 8 e r y t h r o c y t e s w as v i r t u a l l y a b s e n t a m o n g t h e b o t t o m l a y e r cells. O n t h e o t h e r h a n d o n l y a b o u t 2 / 3 o f t h e i n t e r p h a s e l a y e r cells E A C t e s t e d b o u n d m o r e t h a n 8 r e d cells. M o s t o f t h e o t h e r 1 / 3 o f t h e s e cells w e r e w i t h o u t o r h a d o n l y u p t o 2 r e d cells. W i t h r e s p e c t t o b i n d i n g o f 3 - - 5 e r y t h r o c y t e s in E A C t e s t s n o c l e a r c u t d e c i s i o n c a n b e m a d e w h e t h e r s u c h c e l ls a r e p o s i t i v e o r w h e t h e r t h e a s s o c i a t i o n w i t h r e d c e l l s is n o n - s p e c i f i c . T h e y w e r e f o u n d in t h e u n s e p a r a t e d as w e l l as in t h e b o t t o m layer and i n t e r p h a s e layer p o p u l a t i o n s . R o s e t t e f o r m a t i o n with H E A r e s e m b l e s r o s e t t i n g w i t h E A C . T h e f i g u r e s a r e a b o u t t h e s a m e in all cell p o p u l a t i o n s . A s w i t h E A C , o n l y b i n d i n g o f m o r e t h a n 5 r e d cel l s s e e m s t o b e s p e c i f i c f o r F~ r e c e p t o r s . W h e n r e s u l t s w i t h s m e a r s a n d w i t h w e t p r e p a r a t i o n s w e r e c o m p a r e d , in

378

AET

100-

EAC

HEA

80. 60~

A

4o~ i

20

0

0 0 0 Q.

2

4

6 at8

0

2

4

6 1"8

0

2

4

6 k8

1007 80. 60, 40. 20

_ill 0

2

4

6 ~8

erythrocytes

0

B 2

attached

4

6 ~'8

0

2

4

6 k8

to lymphocytes

Fig. 4. R o s e t t e f o r m a t i o n by t h e b l o o d l y m p h o c y t e s of a p a t i e n t with severe c o m b i n e d i m m u n o d e f i c i e n c y t)efore (A) a n d 6 weeks a f t e r h o n e m a r r o w t r a n s p l a n t a t i o n d u r i n g a suba c u t e graft versus h o s t r e a c t i o n (B).

the wet preparations a shift towards cells with lower numbers of attached r e d c e l l s w a s o b s e r v e d . T h i s e f f e c t w a s e s p e c i a l l y o b v i o u s w i t h E- a n d E A C - RFC and was more pronounced when the reading time using wet preparations was prolonged.

TABLE 2 P e r c e n t cells f o r m i n g r o s e t t e s with A E T t r e a t e d S R B C ( A E T ) a n d S R B C c o a t e d with IgM a n t i b o d i e s a n d c o m p l e m e n t ( E A C ) w h e n rosette f o r m i n g cells ( R F C ) are d e f i n e d as ceils b i n d i n g at least 2, 3, 4, or 5 e r y t h r o c y t e s . Exp. *

N u m b e r of a t t a c h e d e r y t h o c y t e s d e f i n i n g a R F C 32

11

33

34

~>5

AET EAC

88.0 40.8

86.4 28.0

85.8 17.4

83.6 16.3

both

128.8

114.4

103.2

98.9

AET EAC

82.2 33.0

77.8 26.6

75.2 25.0

73.8 23.8

both

115.2

104.4

100.2

97.6

379 unseparated 1OO :

60

40 !

40 , _ 0

2

4

6

cells

60 40 ~ 0

20 2

4

6

>8

100

80

80

60

60

60

40

40

40

20

20

20

e',

0

2

4

0

6 >8

2

4

6

>8

80

80

80

60

40

60 40

60 i 40

20

~o 2

2

4

6

>=8

0

2

4

6

>8

0

2

4

6>8

cells of IL after removal of A E T - SRBC 100 100 ]

,ooL 0

0

BL after incubation with A E T - S R B C 100

~oo 1 ID

O

20 ]

:>8

of

100

t

60

20

U

cells

1OO ,

4

6

~ 0

_->8

number

~o t 2

4

6

of erythrocytes

-->s

attached to lymphocytes

Fig. 5. Histograms o f rosette formation by unseparated blood l y m p h o c y t e s , and by cells

recovered from the b o t t o m layer (BL) and the interphase layer (IL) after i n c u b a t i o n with A E T treated SRBC and Ficoll gradient centrifugation.

In table 2 the total numbers o f RFC using A E T and EAC rosette formation as indicators for T and B cells are given on the basis o f different definitions o f a cell as RFC. CONCLUSIONS

The results d e m o n s t r a t e that the m e t h o d o f rosette evaluation on smears described solves p r o b l e m s p o s e d by w e t preparations. Provided the c y t o c e n trifuge smears are carefully prepared so as to p r o d u c e a thin even distribution o f cells, results are reproducible. With a c y t o c e n t r i f u g e , 12 specimens can be analysed at the same time under identical incubation c o n d i t i o n s . The m e t h o d can be applied to observation o f w e a k as well as strong rosettes. The preparations m a y be stored and rosette forming cells c o u n t e d later. E x c e l l e n t d o c u m e n t a t i o n is possible. In a relative short time as m a n y as a t h o u s a n d

380

cells can be scored w i t h o u t the loss o f attached red cells that m a y occur with w e t preparations. Results m a y be recorded as histograms, which m a y be i m p o r t a n t w h e n different incubation c o n d i t i o n s are c o m p a r e d , or w h e n l y m p h o i d cells in tissue cultures or in disease are analysed. The m e t h o d s described m a k e rosette e n u m e r a t i o n s more reliable, c o m p a r a b l e and suitable for wider use. REFERENCES Bianco, C., R. Patrick and V. Nussenzweig, 1970, d. Exp. Med. 132, 702. B0yum, A., 1968, Scan. J. Clin. I,ab. Invest. 21, Suppl. 97, 77. FaRuet, G.B., 197-1, Biomedicine 21, 153. Fr01and, S.S., F. Wish)ff and T.E. Michaelsen, 1974, Inl. Arch. Allergy 47, 1 24. Goldmann, S., D. Niethammcr, t{.J. tiaas, M. Dietrich, tt.-D. Flad, G. Slein, 1976, Z. Immun.-Forsch. in press. Jondal, M., G. Holm and H. Wigzell, 1972, J. Exp. Med. 136, 207. Kapland, M.E. and C. Clark, 197-I, J. hnmun. Methods 5, 131. Ross, G.D.. M.E. Rabellino, M.d. Polley and H.M. Grey, 1973, J. Clin. Invest. 52, 377. Weiner, M.S., C. Bianco and V. Nussenwcig, 1973, Blood 42, 939. Wybran, J., M.C. Carr and t|.H. Fudenberg, 1972, 3. Clin. Invest. 51, 2537. Yam, L.T., C.Y. Li and W.l[. Crosby, 1971, Am. d. Clin. Pathol. 55, 283.