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Automated counting of T cell rosettes
Journal oflmmunological Methods, 10 (1976) 2 6 1 - - 2 7 0
261
© N o r t h - H o l l a n d Publishing C o m p a n y , A m s t e r d a m -- Printed in The Netherlands
AUTOMATED
COUNTING OF T CELL ROSETTES *
A R T H U R A. V A N D E N B A R K 1,2, D E N I S R. B U R G E R 1.2, and R. M A R K V E T T O 1,3
1Surgical Research, Veterans Administration Hospital, Portland, Oregon 97207, U.S.A.; 2 Department of Microbiology and Immunology, University of Oregon Health Sciences Center, Portland, Oregon 97201, U.S.A.; 3 Department of Surgery, University of Oregon Health Sciences Center, Portland, Oregon 97201, U.S.A. (Received 14 August 1975, accepted 9 S e p t e m b e r 1975)
H u m a n T cell rosettes were e n u m e r a t e d using an a u t o m a t e d particle counter, the Bio/ Physics C y t o g r a f 6300A. An electronic oscilloscope representation of particle absorbance and scatter of a focused laser beam allows the separation and e n u m e r a t i o n of b o t h rosetted and non-rosetted l y m p h o c y t e s . R e p e a t e d C y t o g r a f sampling of a single rosette preparation gave highly reproducible results, and sampling from replicate tubes p r o d u c e d the same degree of variation as microscopic analysis. T cell rosettes prepared f r o m 27 volunteers and c o m p a r e d by b o t h m e t h o d s of q u a n t i t a t i o n s h o w e d a high degree of correlation. This m e t h o d can objectively measure at least 100 times as m a n y cells for their rosetteforming capability as the tedious microscopic technique.
INTRODUCTION
The formation of sheep erythrocyte rosettes is a widely used procedure to identify human T cells (Coombs et al., 1970; Fr¢land, 1972; Jondal et al., 1972; Wybran et al., 1972; Bach, 1973; Wortis et al., 1973). Rosette forming cells (RFC) have usually been detected by microscopic observation using a hemocytometer. This process is time consuming, is limited by sampling error, and is dependent on the patience and the expertise of the investigator. This report describes the use of the Bio/Physics model 6300A Cytograf for impartially quantitating large numbers of T cell rosettes with improved efficiency over conventional techniques. MATERIALS AND METHODS
L y m p h o c y te prepara tion
Five ml heparinized (8 U/ml) human venous blood was diluted with 5 ml culture medium (RPMI 1640 containing 25 mM Hepes buffer and antibiot* This research was s u p p o r t e d by project n u m b e r 8958-01 f r o m the Veterans Administration Hospital, Portland, Oregon 97207.
262 ics), layered over 5 ml Ficoll--Isopaque mixture, and centrifuged at 4 0 0 g for 30 min (Harris and Ukaejiofo, 1969). The mononuclear rich fraction at the media-separation mixture interface was removed, washed 2 times in media (sedimented at 200 g for 10 min), and resuspended at a concentration of 4 X 106 cells/ml in media. Differential counts of Wright-Geimsa stained smears indicated that these preparations contained >95% lymphocytes.
Rosette formation For rosette preparation 0.25 ml of sheep erythrocytes (washed in media and resuspended at 3 X 107 RBC/ml) and 0.25 ml of each l y m p h o c y t e suspension were added in duplicate or triplicate to plastic tubes (Falcon Plastics, #2005). These tubes were incubated at 37°C for 5 min, packed at 200 g for 5 min (Jondal et al., 1972), and were stored overnight at 5°C. The cells were resuspended in a total of 3 ml media for counting by gently inverting each tube 20 times.
Rosette quantitation Microscopic: The rosette suspension was added to a h e m o c y t o m e t e r and observed at 100X for RFC and non-rosetted lymphocytes. L y m p h o c y t e s which bound three or more sheep erythrocytes were considered RFC. The cells in 0.1 mm 3 were counted (usually between 200--250 cells) from duplicate or triplicate tubes from each l y m p h o c y t e donor. Percent rosettes = (RFC X 100)/total lymphocytes. Bio/Physics Cytograf (model 6300A), Instrumentation: Particles added to the Cytograf are discriminated on the basis of both forward angle scatter and axial light loss (absorbance) of a focused laser beam (fig. 1). An electronic signal representing these two characteristics of each cell is projected onto an oscilloscope screen (X axis, scatter; Y axis, absorbance). The oscilloscope screen stores and displays these signals which allows visualization of the subpopulations of particles. Since larger particles are recorded further from the X--Y origin than smaller particles, the rosettes are displayed on the screen as a discrete population separated from the non-rosetted lymphocytes and sheep erythrocytes. The Cytograf quantitates the total number of particles on the screen as well as the particles within any preselected area: in this manner it is possible to electronically delineate a selected area which includes rosettes and a few overlapping large lymphocytes but which excludes >95% of the non-rosetted lymphocytes and all sheep erythrocytes. Procedure: 0.5 ml of the rosette suspension was introduced into the counting chamber of the Cytograf, and the total number of particles in the selected window (rosettes plus overlapping non-rosetted cells) were recorded. In initial studies, this procedure was repeated 10 times per tube to assess machine and sampling variability. The erythrocytes in the same sample tube were then lysed with a saponin solution (ZAP, Coulter Products), leaving an
263
Fig. 1. Diagrammatic representation of Cytograf 6300A. The laser light source (A) illuminates the cells (B). Light scatter or absorbance is detected by photosensor (C) and is displayed as a two-dimensional dot pattern (X axis = scatter; Y axis = absorption) on an oscilloscope screen (D). Cell populations include, 1)sheep erythrocytes, 2)lymphocytes, and 3) rosetted lymphocytes. The electronic threshold (T) eliminates debris from total cell counts (E), and electronic settings (p, r, and s) delineate the selected count area (F). The rosette determination is done in two parts; 1 ) the rosetted preparation is counted but only the particles in the selected window (rosettes and overlapping large cells) are recorded, 2) the sheep erythrocytes are lysed and the preparation is counted a second time. Both the total count (lymphocytes) and the selected count (overlapping large cells) tallies are recorded. The percentage of rosettes is determined by subtracting the overlapping large cells from the combined rosette-overlapping large cell count, and dividing the difference by the total lymphocyte tally. e r y t h r o c y t e free l y m p h o c y t e suspension. One-half ml replicates o f the e r y t h r o c y t e free s u s p e n s i o n were c o u n t e d in the C y t o g r a f and b o t h the t o t a l n u m ber o f particles (total l y m p h o c y t e s ) , and the n u m b e r o f particles in the selected c o u n t w i n d o w (overlapping n o n - r o s e t t e d cells) were r e c o r d e d . T h e perc e n t rosettes were d e t e r m i n e d as follows: t o t a l particles in r o s e t t e w i n d o w - - o v e r l a p p i n g n o n - r o s e t t e d cells total l y m p h o c y t e s RESULTS T h e cell p o p u l a t i o n s o b s e r v e d w h e n c o u n t i n g r o s e t t e d h u m a n l y m p h o c y t e s on t h e C y t o g r a f 6 3 0 0 A are s h o w n in fig. 2. The r o s e t t e suspension {containing rosettes, n o n - r o s e t t e d l y m p h o c y t e s and e r y t h r o c y t e s ) s h o w n in fig. 2a gives a p a t t e r n clearly distinguishable f r o m t h e m a j o r i t y o f t h e iso-
264
jr
~,
zZ// ~o
Scatter Fig. 2. P h o t o g r a p h s o f C y t o g r a f r e p r e s e n t a t i o n of, a) s u s p e n s i o n o f r o s e t t e f o r m i n g cells, l y m p h o c y t e s , and s h e e p red b l o o d cells; a n d b) same s u s p e n s i o n a f t e r lysis o f all SRBC. X axis = s c a t t e r ; Y axis = a b s o r b a n c e ; d o t t e d lines indicate selected c o u n t w i n d o w .
TABLE 1 Setting o f s e l e c t e d c o u n t w i n d o w o f C y t o g r a f 6 3 0 0 A for r o s e t t e d i s c r i m i n a t i o n . p setting a
Percent rosettes M i c r o s c o p i c analysis b 44 46
50
53
59
70 56 51 48 45
75 63 55 50 48
70 64 59 56 51
C y t o g r a f analysis b 32 33 34 35 36
67 54 43 34 29
64 59 50 43 41
a p s e t t i n g d e t e r m i n e s t h e l o w e r t h r e s h o l d b e t w e e n the r o s e t t e s and t h e n o n - r o s e t t e d lymp h o c y t e s , r, and s settings were arbitrarily set to include all particles (see fig. 1). b Each value r e p r e s e n t s m e a n o f d u p l i c a t e d e t e r m i n a t i o n s .
265 TABLE 2 Sensitivity of the Cytograf 6300A a. Lymphocytes/0.5 ml
Percent rosettes (Cytograf)
4500 8000 16000 34O00 67000 135000
63 62 63 63 68 70
-+ 1 -+ 2 +1 +3 +1b +3b
a Duplicate Cytograf determinations were carried out at various concentrations of a preparation containing 59 _+2% rosettes as determined by microscopy. b Values are significantly different from microscopically determined value.
lated l y m p h o c y t e s ( Z A P - t r e a t e d r o s e t t e suspension) (fig. 2b). T h e s e p a r a t i o n o f r o s e t t e d and n o n - r o s e t t e d l y m p h o c y t e s is indicated b y the d o t t e d line in fig. 2 which delineates the selected c o u n t w i n d o w . In this p a r t i c u l a r preparation, t h e r e were 8 3 6 4 particles in the selected c o u n t w i n d o w in fig. 2a, 287 overlapping n o n - r o s e t t e d l y m p h o c y t e s in the selected c o u n t w i n d o w in fig. 2b, and a t o t a l o f 1 6 6 1 6 l y m p h o c y t e s (fig. 2b). This calculates as 48.4% rosettes which is the same p e r c e n t d e t e r m i n e d b y m i c r o s c o p i c e x a m i n a t i o n . The margins o f the selected c o u n t w i n d o w are set b y e l e c t r o n i c c o n t r o l s p, r, and s (fig. 1). T h e r and s c o n t r o l s were set arbitrarily, b u t the p c o n t r o l setting which separates the r o s e t t e d f r o m the n o n - r o s e t t e d particles was d e t e r m i n e d e x p e r i m e n t a l l y f r o m five p r e p a r a t i o n s ranging f r o m 4 4 - - 5 9 % rosettes (microscopically d e t e r m i n e d ) . T h e results f r o m this e x p e r i m e n t (table 1) indicate t h a t a p setting o f 34 m o s t closely a p p r o x i m a t e s the p e r c e n t rosettes previously d e t e r m i n e d b y the m i c r o s c o p i c m e t h o d . Based o n t h e d a t a in table 1 a p setting o f 34 was s u b s e q u e n t l y utilized in all r o s e t t e d e t e r m i n a t i o n s (with the e x c e p t i o n o f an occasional sample which had an u n u s u a l l y low percentage o f large l y m p h o c y t e s . See discussion below). In o r d e r to d e t e r m i n e t h e range o f cell c o n c e n t r a t i o n s which c o u l d be c o u n t e d in the C y t o g r a f 6 3 0 0 A , various dilutions o f a r o s e t t e suspension were c o u n t e d and c o m p a r e d t o a m i c r o s c o p i c a l l y a n a l y z e d r o s e t t e preparation (table 2). T o t a l l y m p h o c y t e c o u n t s as high as 3 4 , 0 0 0 per 0.5 ml consist e n t l y gave 6 2 - - 6 3 % rosettes ( n o t significantly d i f f e r e n t f r o m 59% determ i n e d m i c r o s c o p i c a l l y ) . H o w e v e r , a t w o - f o l d higher c o n c e n t r a t i o n o f lymp h o c y t e s increased t h e C y t o g r a f p e r c e n t a g e o f r o s e t t e s t o 67.5%, and a fourfold increase in l y m p h o c y t e c o n c e n t r a t i o n f u r t h e r raised the p e r c e n t a g e o f r o s e t t e s t o 69.5% ( b o t h o f the latter d e t e r m i n a t i o n s were significantly higher t h a n t h e m i c r o s c o p i c a l l y d e t e r m i n e d value). T o t a l l y m p h o c y t e c o u n t s as low as 4 5 0 0 per 0.5 ml gave 63% r o s e t t e s ( C y t o g r a f ) which was n o t d i f f e r e n t f r o m t h e m i c r o s c o p i c a l l y d e t e r m i n e d value. Thus, in s u b s e q u e n t r o s e t t e det e r m i n a t i o n s , 3 4 , 0 0 0 or f e w e r l y m p h o c y t e s were c o u n t e d .
266
In order to assess the reproducibility in counting either 1) the rosette preparation or 2) the e r y t h r o c y t e free preparation, ten sequential counts were made on each suspension. The rosette suspension contained 17781 rosettes with standard deviation of 564 (S.D. = 3.2% of mean), whereas the erythr0cyte free suspension had 26389 ± 760 (S.D. = 2.9% of mean) total lymphocytes, and 1174 i 58 (S.D. = 4.9% of mean) overlapping large cells that were detected in the selected c o u n t window. In a similar experiment designed to test the reproducibility of measuring percent rosettes in the same tube, both steps in the rosette determination (measurement of rosette suspension followed by measurement of the erythrocyte free suspension) were carried o u t in paired fashion in quadruplicate (table 3). Each complete rosette determination led to a separate percentage of rosettes from the same tube. This procedure allowed an analysis of variance among 4 samples drawn from the same tube which could be compared to the variance among 4 samples (also from the same tube) determined microscopically by three qualified personnel (table 3). The results of this comTABLE 3 C o m p a r i s o n o f r e p l i c a t e C y t o g r a f vs m i c r o s c o p i c r o s e t t e d e t e r m i n a t i o n s . Rosettes
14948 14602 14716 14666
Overlapping large cells 894 832 862 857
C y t o g r a f analysis Total lymphocytes
% Rosettes
23707 22864 23838 23975
59.3 60.2 58.1 57.6
X ± SD
58.8 ± 1.2 Observer
Total rosettes
142 132 132 159
M i c r o s c o p i c analysis Total lymphocytes
% Rosettes
251 222 225 270
56.6 59.5 58.7 58.9
X ± SD
58.4±1.3 153 154 142 132
270 275 234 221
56.7 56.0 60.7 59.7
58.3±2.3 150 165 158 162
260 278 268 271
57.7 59.4 59.0 59.8 59.0±0.9
267 TABLE 4 Comparison of Cytograf and microscopic f r o m 27 v o l u n t e e r s .
determination
C y t o g r a f R F C (% -+ S D )
M i c r o s c o p e R F C (% -+ S D )
52+2 69+3 61 + 1 55 + 6 41_+6 59 -+ 11 31-+1 43-+8 48-+4 46-+2 52 -+ 5 42 + 1 46-+4 50-+1 57 -+ 6 4 5 -+ 1 42-+5 53+1
59+2 63+1 61 + 2 60 + 6 49+6 54 + 4 34-+4 61-+5 46-+8 62+7 4 8 -+ 3 3 4 -+ 7 49-+1 56-+15 6 0 -+ 5 4 6 -+ 2 55+6 49-+6
51-+4
53-+1
4 8 -+ 4 71-+13 59 -+ 1 4 3 -+ 1 5 0 -+ 2 51 + 4 55 -+ 2 59 -+ 1
51 -+ 6 68-+4 59 + 2 4 4 -+ 2 4 6 -+ 3 50 -+ 2 53 + 4 5 9 -+ 2
o f r o s e t t e - f o r m i n g cells ( R F C )
a a
a a
a
a Cytograf and microscopically determined
m e a n s a r e s i g n i f i c a n t l y d i f f e r e n t ( P <: 0 . 0 5 ) .
parison show that the standard deviations of replicate determinations on the same t u b e by the t w o methods are similar (1.2% for Cytograf versus an average of 1.5% for microscopic determination). However, the results obtained by the Cytograf analysis are based on a much larger sample population than the microscopic evaluation. The Cytograf produced replicate analysis of approximately 25,000 l y m p h o c y t e s o u t of a total of 106 l y m p h o c y t e s in each tube, whereas the microscopic technique sampled approximately 250 cells. In order to compare the percent rosette obtained by each method, both Cytograf and microscopic rosette determinations were carried out on replicate l y m p h o c y t e suspensions from 27 donors (table 4). The mean % rosettes + standard deviation (S.D.) obtained by the t w o techniques for each donor were consistently similar (only 5 of the 27 pairs had significant differences). The rosette values obtained by each method are shown in fig. 3 (X axis, mi-
268 LINEAR PLOTOF CYTOGRAFVERSUS MICROSCOPIC DETERMINATION OF PERCENTROSETTE FORMING CELLS (RFC) FROM 27 VOLUNTEERS I0o
80
/// ///////
'~
e e
~ 4o ~:
20
2~o
4'0
o'o
B'o
,~o
% RFC (Microscopic Anolysis)
Fig. 3. Data from 27 volunteers comparing percent rosette forming cells (RFC) obtained by Cytograf 6300A versus microscopic analysis. Points have correlation coefficient (R) of 0.72 for dotted line (Y = 11.23 + 0.754X). Solid line represents theoretical 1 to 1 correlation of the two parameters. c r o s c o p i c analysis; Y axis, C y t o g r a f analysis). T h e d o t t e d line (Y = 11.23 + 0 . 7 5 4 X ) r e p r e s e n t s the line o f best fit f o r the 27 p o i n t s and has a c o r r e l a t i o n c o e f f i c i e n t o f 0.72 ( p e r f e c t c o r r e l a t i o n = 1.0) which indicates a highly significant degree o f c o r r e l a t i o n o f the t w o m e t h o d s (P < 0.01). DISCUSSION T h e p r o c e d u r e f o r q u a n t i t a t i n g T cells b y measuring sheep e r y t h r o c y t e rosettes is simple and specific, b u t the discrimination o f r o s e t t e s f r o m nonr o s e t t e d l y m p h o c y t e s o f t e n requires t e d i o u s m i c r o s c o p i c e x a m i n a t i o n o f a limited sampling o f cells. This r e p o r t describes the use o f the C y t o g r a f 6 3 0 0 A (Bio/Physics) t o a u t o m a t i c a l l y q u a n t i t a t e large n u m b e r s o f r o s e t t e - f o r m i n g cells. We have f o u n d t h a t this i n s t r u m e n t saves considerable t i m e and e f f o r t w h e n d e t e r m i n i n g R F C in multiple samples o n a r o u t i n e basis. The C y t o g r a f 6 3 0 0 A analysis samples a p p r o x . 100 times the n u m b e r o f cells and quantitates R F C r e p r o d u c i b l y with g o o d c o r r e l a t i o n t o m i c r o s c o p i c a l l y d e t e r m i n e d R F C percents. T h e d a t a in table 1 indicate t h a t a single c o n t r o l setting 09 = 34) can accur a t e l y a p p r o x i m a t e t h e p e r c e n t R F C for five representative individuals over a range o f m i c r o s c o p i c a l l y d e t e r m i n e d values (44--59%). T h e size distrib u t i o n o f l y m p h o c y t e s f r o m these individuals ( w h o include 2 c a n c e r p a t i e n t s and 3 n o r m a l v o l u n t e e r s ) shows considerable visual variation (on the oscilloscope); h o w e v e r , t h e p e r c e n t o f n o n - r o s e t t e d overlapping cells t h a t are inc l u d e d in the selected c o u n t (rosette) w i n d o w o n l y ranges f r o m 2.1--3.1%. It is likely t h a t a C y t o g r a f analysis o f R F C f r o m an individual with a signifi-
269 cantly smaller l y m p h o c y t e size distribution would be inaccurate, since many of the smaller rosettes might be deleted from the selected window (with a p = 34 setting). Indeed, one of our initial determinations showed only 0.7% overlapping cells, and correspondingly showed a large discrepancy in Cytograf vs microscopic RFC analysis (46 vs 62%). Individuals with less than 1.5% overlapping cells are currently being retested with the p control set to include an arbitrary 2.5% overlapping cells. Overlapping cell percents in the higher ranges (as high as 8.5%) did not result in significant differences in Cytograf and microscopic RFC values. The data presented in table 2 indicate that a two-fold increase in the number of cells counted above 35,000 results in an erroneous Cytograf determination of % RFC. This is probably the result of coincident particle counting, e.g. the Cytograf sensors detect two or more closely positioned smaller particles as a single large particle. The effects of coincidence increases the number of particles in the selected count window and thus increases the % RFC as observed. The Cytograf 6300A gives highly reproducible results when sequential replicate determinations are carried out first on the rosette preparation and then on the ZAP treated suspension. This data is included for practical reasons only. Replicate determinations performed on rosettes from several individuals is slowed by the thorough cleaning of the sample chamber that is required after each ZAP treated suspension is counted. This data indicates that the operator can complete the rosette count for all the samples to be tested (with minimal cleanings) and then count all the ZAP treated suspensions (with minimal intermittent cleaning). Rosette analysis performed using alternating rosetted and ZAP treated preparations (with thorough washing in between) allows statistical analysis of replicate determinations of the same sample which can be compared to replicate microscopic analysis of RFC% (table 3). This data indicates that the variance of the Cytograf analysis done in this manner is comparable to the variance in microscopic analysis performed by three experienced personnel. The two step Cytograf procedure outlined above allows quantitation of total lymphocytes, large lymphocytes, and RFC from the same tube. Attempts to measure total and large lymphocytes in a separate tube from the RFC (thus avoiding the lytic agent) have resulted in increased sample variation. Table 4 shows that the mean RFC percents obtained by Cytograf analysis of replicate tubes from 27 volunteers (including cancer patients and normal individuals) are very similar to microscopically determined values. The variation in RFC% from replicate tubes is reflected by the mean of the standard deviation, which is similar for the two methods (Cytograf, 3.9; microscope, 4.3). The plot of the data in table 4 (fig. 3) shows a highly significant correlation (P ~ 0.01) in % RFC determined by the two analyses (correlation coefficient R of the points for the line of best fit = 0.72). This correlation is represented by 22 of 27 samples t h a t are n o t different statistically, and 5 of 27 that are different. In one of the latter samples a predominantly small
270 l y m p h o c y t e p o p u l a t i o n biased the C y t o g r a f result (discussed above), while in the r e m a i n d e r , the d i s c r e p a n c y m i g h t be a t t r i b u t e d t o subjective errors associated with m i c r o s c o p i c evaluation. T h e C y t o g r a f t e c h n i q u e d e s c r i b e d above is easily a d a p t e d to d e t e r m i n e p e r c e n t o f live cells t h a t f o r m rosettes. T h e o n l y steps t h a t n e e d be a d d e d are, 1) t o s u p p l e m e n t the Z A P e d l y m p h o c y t e suspension with a vital d y e ( T r y p a n Blue), and 2) t o q u a n t i t a t e p e r c e n t viable l y m p h o c y t e s on the C y t o g r a f b y t h e m e t h o d o f Wilson et al. (1973). P e r c e n t R F C w o u l d t h a n be calculated b y using viable instead o f t o t a l l y m p h o c y t e s . ACKNOWLEDGEMENTS We are g r a t e f u l f o r the assistance o f H a t s u m i Park and Patricia Finke.
REFERENCES Bach, J.F., 1973, Transplant. Rev. 16, 196. Coombs, R.R.A., B.W. Gurner, A.B. Wilson, G. Holm and B. Lindgren, 1970, Int. Arch. Allergy 39,658. Fr¢land, S.S., 1972, Scand. J. Immunol. 1,269. Harris, R. and E.O. Ukaejiofo, 1969, Lancet 2,327. Jondal, M., G. Holm and H. Wigzell, 1972, J. Exp. Med. 136,207. Papamichail, M., E.J. Holborow, H.I. Keith et al., 1972, Lancet 2, 64. Silveira, N.P.A., N.F. Mendes and M.E.A. Tolnai, 1972, J. Immunol. 108, 1456. Wilson, M., J. Dardano and H. Rothschild, 1973, Transplantation 16,408. Wortis, H.H., A.G. Cooper and M.C. Brown, 1973, Nature New Biol. 243,109. Wybran, J., M.C. Carr and H.H. Fudenberg, 1972, J. Clin. Invest. 51, 2537.
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© N o r t h - H o l l a n d Publishing C o m p a n y , A m s t e r d a m -- Printed in The Netherlands
AUTOMATED
COUNTING OF T CELL ROSETTES *
A R T H U R A. V A N D E N B A R K 1,2, D E N I S R. B U R G E R 1.2, and R. M A R K V E T T O 1,3
1Surgical Research, Veterans Administration Hospital, Portland, Oregon 97207, U.S.A.; 2 Department of Microbiology and Immunology, University of Oregon Health Sciences Center, Portland, Oregon 97201, U.S.A.; 3 Department of Surgery, University of Oregon Health Sciences Center, Portland, Oregon 97201, U.S.A. (Received 14 August 1975, accepted 9 S e p t e m b e r 1975)
H u m a n T cell rosettes were e n u m e r a t e d using an a u t o m a t e d particle counter, the Bio/ Physics C y t o g r a f 6300A. An electronic oscilloscope representation of particle absorbance and scatter of a focused laser beam allows the separation and e n u m e r a t i o n of b o t h rosetted and non-rosetted l y m p h o c y t e s . R e p e a t e d C y t o g r a f sampling of a single rosette preparation gave highly reproducible results, and sampling from replicate tubes p r o d u c e d the same degree of variation as microscopic analysis. T cell rosettes prepared f r o m 27 volunteers and c o m p a r e d by b o t h m e t h o d s of q u a n t i t a t i o n s h o w e d a high degree of correlation. This m e t h o d can objectively measure at least 100 times as m a n y cells for their rosetteforming capability as the tedious microscopic technique.
INTRODUCTION
The formation of sheep erythrocyte rosettes is a widely used procedure to identify human T cells (Coombs et al., 1970; Fr¢land, 1972; Jondal et al., 1972; Wybran et al., 1972; Bach, 1973; Wortis et al., 1973). Rosette forming cells (RFC) have usually been detected by microscopic observation using a hemocytometer. This process is time consuming, is limited by sampling error, and is dependent on the patience and the expertise of the investigator. This report describes the use of the Bio/Physics model 6300A Cytograf for impartially quantitating large numbers of T cell rosettes with improved efficiency over conventional techniques. MATERIALS AND METHODS
L y m p h o c y te prepara tion
Five ml heparinized (8 U/ml) human venous blood was diluted with 5 ml culture medium (RPMI 1640 containing 25 mM Hepes buffer and antibiot* This research was s u p p o r t e d by project n u m b e r 8958-01 f r o m the Veterans Administration Hospital, Portland, Oregon 97207.
262 ics), layered over 5 ml Ficoll--Isopaque mixture, and centrifuged at 4 0 0 g for 30 min (Harris and Ukaejiofo, 1969). The mononuclear rich fraction at the media-separation mixture interface was removed, washed 2 times in media (sedimented at 200 g for 10 min), and resuspended at a concentration of 4 X 106 cells/ml in media. Differential counts of Wright-Geimsa stained smears indicated that these preparations contained >95% lymphocytes.
Rosette formation For rosette preparation 0.25 ml of sheep erythrocytes (washed in media and resuspended at 3 X 107 RBC/ml) and 0.25 ml of each l y m p h o c y t e suspension were added in duplicate or triplicate to plastic tubes (Falcon Plastics, #2005). These tubes were incubated at 37°C for 5 min, packed at 200 g for 5 min (Jondal et al., 1972), and were stored overnight at 5°C. The cells were resuspended in a total of 3 ml media for counting by gently inverting each tube 20 times.
Rosette quantitation Microscopic: The rosette suspension was added to a h e m o c y t o m e t e r and observed at 100X for RFC and non-rosetted lymphocytes. L y m p h o c y t e s which bound three or more sheep erythrocytes were considered RFC. The cells in 0.1 mm 3 were counted (usually between 200--250 cells) from duplicate or triplicate tubes from each l y m p h o c y t e donor. Percent rosettes = (RFC X 100)/total lymphocytes. Bio/Physics Cytograf (model 6300A), Instrumentation: Particles added to the Cytograf are discriminated on the basis of both forward angle scatter and axial light loss (absorbance) of a focused laser beam (fig. 1). An electronic signal representing these two characteristics of each cell is projected onto an oscilloscope screen (X axis, scatter; Y axis, absorbance). The oscilloscope screen stores and displays these signals which allows visualization of the subpopulations of particles. Since larger particles are recorded further from the X--Y origin than smaller particles, the rosettes are displayed on the screen as a discrete population separated from the non-rosetted lymphocytes and sheep erythrocytes. The Cytograf quantitates the total number of particles on the screen as well as the particles within any preselected area: in this manner it is possible to electronically delineate a selected area which includes rosettes and a few overlapping large lymphocytes but which excludes >95% of the non-rosetted lymphocytes and all sheep erythrocytes. Procedure: 0.5 ml of the rosette suspension was introduced into the counting chamber of the Cytograf, and the total number of particles in the selected window (rosettes plus overlapping non-rosetted cells) were recorded. In initial studies, this procedure was repeated 10 times per tube to assess machine and sampling variability. The erythrocytes in the same sample tube were then lysed with a saponin solution (ZAP, Coulter Products), leaving an
263
Fig. 1. Diagrammatic representation of Cytograf 6300A. The laser light source (A) illuminates the cells (B). Light scatter or absorbance is detected by photosensor (C) and is displayed as a two-dimensional dot pattern (X axis = scatter; Y axis = absorption) on an oscilloscope screen (D). Cell populations include, 1)sheep erythrocytes, 2)lymphocytes, and 3) rosetted lymphocytes. The electronic threshold (T) eliminates debris from total cell counts (E), and electronic settings (p, r, and s) delineate the selected count area (F). The rosette determination is done in two parts; 1 ) the rosetted preparation is counted but only the particles in the selected window (rosettes and overlapping large cells) are recorded, 2) the sheep erythrocytes are lysed and the preparation is counted a second time. Both the total count (lymphocytes) and the selected count (overlapping large cells) tallies are recorded. The percentage of rosettes is determined by subtracting the overlapping large cells from the combined rosette-overlapping large cell count, and dividing the difference by the total lymphocyte tally. e r y t h r o c y t e free l y m p h o c y t e suspension. One-half ml replicates o f the e r y t h r o c y t e free s u s p e n s i o n were c o u n t e d in the C y t o g r a f and b o t h the t o t a l n u m ber o f particles (total l y m p h o c y t e s ) , and the n u m b e r o f particles in the selected c o u n t w i n d o w (overlapping n o n - r o s e t t e d cells) were r e c o r d e d . T h e perc e n t rosettes were d e t e r m i n e d as follows: t o t a l particles in r o s e t t e w i n d o w - - o v e r l a p p i n g n o n - r o s e t t e d cells total l y m p h o c y t e s RESULTS T h e cell p o p u l a t i o n s o b s e r v e d w h e n c o u n t i n g r o s e t t e d h u m a n l y m p h o c y t e s on t h e C y t o g r a f 6 3 0 0 A are s h o w n in fig. 2. The r o s e t t e suspension {containing rosettes, n o n - r o s e t t e d l y m p h o c y t e s and e r y t h r o c y t e s ) s h o w n in fig. 2a gives a p a t t e r n clearly distinguishable f r o m t h e m a j o r i t y o f t h e iso-
264
jr
~,
zZ// ~o
Scatter Fig. 2. P h o t o g r a p h s o f C y t o g r a f r e p r e s e n t a t i o n of, a) s u s p e n s i o n o f r o s e t t e f o r m i n g cells, l y m p h o c y t e s , and s h e e p red b l o o d cells; a n d b) same s u s p e n s i o n a f t e r lysis o f all SRBC. X axis = s c a t t e r ; Y axis = a b s o r b a n c e ; d o t t e d lines indicate selected c o u n t w i n d o w .
TABLE 1 Setting o f s e l e c t e d c o u n t w i n d o w o f C y t o g r a f 6 3 0 0 A for r o s e t t e d i s c r i m i n a t i o n . p setting a
Percent rosettes M i c r o s c o p i c analysis b 44 46
50
53
59
70 56 51 48 45
75 63 55 50 48
70 64 59 56 51
C y t o g r a f analysis b 32 33 34 35 36
67 54 43 34 29
64 59 50 43 41
a p s e t t i n g d e t e r m i n e s t h e l o w e r t h r e s h o l d b e t w e e n the r o s e t t e s and t h e n o n - r o s e t t e d lymp h o c y t e s , r, and s settings were arbitrarily set to include all particles (see fig. 1). b Each value r e p r e s e n t s m e a n o f d u p l i c a t e d e t e r m i n a t i o n s .
265 TABLE 2 Sensitivity of the Cytograf 6300A a. Lymphocytes/0.5 ml
Percent rosettes (Cytograf)
4500 8000 16000 34O00 67000 135000
63 62 63 63 68 70
-+ 1 -+ 2 +1 +3 +1b +3b
a Duplicate Cytograf determinations were carried out at various concentrations of a preparation containing 59 _+2% rosettes as determined by microscopy. b Values are significantly different from microscopically determined value.
lated l y m p h o c y t e s ( Z A P - t r e a t e d r o s e t t e suspension) (fig. 2b). T h e s e p a r a t i o n o f r o s e t t e d and n o n - r o s e t t e d l y m p h o c y t e s is indicated b y the d o t t e d line in fig. 2 which delineates the selected c o u n t w i n d o w . In this p a r t i c u l a r preparation, t h e r e were 8 3 6 4 particles in the selected c o u n t w i n d o w in fig. 2a, 287 overlapping n o n - r o s e t t e d l y m p h o c y t e s in the selected c o u n t w i n d o w in fig. 2b, and a t o t a l o f 1 6 6 1 6 l y m p h o c y t e s (fig. 2b). This calculates as 48.4% rosettes which is the same p e r c e n t d e t e r m i n e d b y m i c r o s c o p i c e x a m i n a t i o n . The margins o f the selected c o u n t w i n d o w are set b y e l e c t r o n i c c o n t r o l s p, r, and s (fig. 1). T h e r and s c o n t r o l s were set arbitrarily, b u t the p c o n t r o l setting which separates the r o s e t t e d f r o m the n o n - r o s e t t e d particles was d e t e r m i n e d e x p e r i m e n t a l l y f r o m five p r e p a r a t i o n s ranging f r o m 4 4 - - 5 9 % rosettes (microscopically d e t e r m i n e d ) . T h e results f r o m this e x p e r i m e n t (table 1) indicate t h a t a p setting o f 34 m o s t closely a p p r o x i m a t e s the p e r c e n t rosettes previously d e t e r m i n e d b y the m i c r o s c o p i c m e t h o d . Based o n t h e d a t a in table 1 a p setting o f 34 was s u b s e q u e n t l y utilized in all r o s e t t e d e t e r m i n a t i o n s (with the e x c e p t i o n o f an occasional sample which had an u n u s u a l l y low percentage o f large l y m p h o c y t e s . See discussion below). In o r d e r to d e t e r m i n e t h e range o f cell c o n c e n t r a t i o n s which c o u l d be c o u n t e d in the C y t o g r a f 6 3 0 0 A , various dilutions o f a r o s e t t e suspension were c o u n t e d and c o m p a r e d t o a m i c r o s c o p i c a l l y a n a l y z e d r o s e t t e preparation (table 2). T o t a l l y m p h o c y t e c o u n t s as high as 3 4 , 0 0 0 per 0.5 ml consist e n t l y gave 6 2 - - 6 3 % rosettes ( n o t significantly d i f f e r e n t f r o m 59% determ i n e d m i c r o s c o p i c a l l y ) . H o w e v e r , a t w o - f o l d higher c o n c e n t r a t i o n o f lymp h o c y t e s increased t h e C y t o g r a f p e r c e n t a g e o f r o s e t t e s t o 67.5%, and a fourfold increase in l y m p h o c y t e c o n c e n t r a t i o n f u r t h e r raised the p e r c e n t a g e o f r o s e t t e s t o 69.5% ( b o t h o f the latter d e t e r m i n a t i o n s were significantly higher t h a n t h e m i c r o s c o p i c a l l y d e t e r m i n e d value). T o t a l l y m p h o c y t e c o u n t s as low as 4 5 0 0 per 0.5 ml gave 63% r o s e t t e s ( C y t o g r a f ) which was n o t d i f f e r e n t f r o m t h e m i c r o s c o p i c a l l y d e t e r m i n e d value. Thus, in s u b s e q u e n t r o s e t t e det e r m i n a t i o n s , 3 4 , 0 0 0 or f e w e r l y m p h o c y t e s were c o u n t e d .
266
In order to assess the reproducibility in counting either 1) the rosette preparation or 2) the e r y t h r o c y t e free preparation, ten sequential counts were made on each suspension. The rosette suspension contained 17781 rosettes with standard deviation of 564 (S.D. = 3.2% of mean), whereas the erythr0cyte free suspension had 26389 ± 760 (S.D. = 2.9% of mean) total lymphocytes, and 1174 i 58 (S.D. = 4.9% of mean) overlapping large cells that were detected in the selected c o u n t window. In a similar experiment designed to test the reproducibility of measuring percent rosettes in the same tube, both steps in the rosette determination (measurement of rosette suspension followed by measurement of the erythrocyte free suspension) were carried o u t in paired fashion in quadruplicate (table 3). Each complete rosette determination led to a separate percentage of rosettes from the same tube. This procedure allowed an analysis of variance among 4 samples drawn from the same tube which could be compared to the variance among 4 samples (also from the same tube) determined microscopically by three qualified personnel (table 3). The results of this comTABLE 3 C o m p a r i s o n o f r e p l i c a t e C y t o g r a f vs m i c r o s c o p i c r o s e t t e d e t e r m i n a t i o n s . Rosettes
14948 14602 14716 14666
Overlapping large cells 894 832 862 857
C y t o g r a f analysis Total lymphocytes
% Rosettes
23707 22864 23838 23975
59.3 60.2 58.1 57.6
X ± SD
58.8 ± 1.2 Observer
Total rosettes
142 132 132 159
M i c r o s c o p i c analysis Total lymphocytes
% Rosettes
251 222 225 270
56.6 59.5 58.7 58.9
X ± SD
58.4±1.3 153 154 142 132
270 275 234 221
56.7 56.0 60.7 59.7
58.3±2.3 150 165 158 162
260 278 268 271
57.7 59.4 59.0 59.8 59.0±0.9
267 TABLE 4 Comparison of Cytograf and microscopic f r o m 27 v o l u n t e e r s .
determination
C y t o g r a f R F C (% -+ S D )
M i c r o s c o p e R F C (% -+ S D )
52+2 69+3 61 + 1 55 + 6 41_+6 59 -+ 11 31-+1 43-+8 48-+4 46-+2 52 -+ 5 42 + 1 46-+4 50-+1 57 -+ 6 4 5 -+ 1 42-+5 53+1
59+2 63+1 61 + 2 60 + 6 49+6 54 + 4 34-+4 61-+5 46-+8 62+7 4 8 -+ 3 3 4 -+ 7 49-+1 56-+15 6 0 -+ 5 4 6 -+ 2 55+6 49-+6
51-+4
53-+1
4 8 -+ 4 71-+13 59 -+ 1 4 3 -+ 1 5 0 -+ 2 51 + 4 55 -+ 2 59 -+ 1
51 -+ 6 68-+4 59 + 2 4 4 -+ 2 4 6 -+ 3 50 -+ 2 53 + 4 5 9 -+ 2
o f r o s e t t e - f o r m i n g cells ( R F C )
a a
a a
a
a Cytograf and microscopically determined
m e a n s a r e s i g n i f i c a n t l y d i f f e r e n t ( P <: 0 . 0 5 ) .
parison show that the standard deviations of replicate determinations on the same t u b e by the t w o methods are similar (1.2% for Cytograf versus an average of 1.5% for microscopic determination). However, the results obtained by the Cytograf analysis are based on a much larger sample population than the microscopic evaluation. The Cytograf produced replicate analysis of approximately 25,000 l y m p h o c y t e s o u t of a total of 106 l y m p h o c y t e s in each tube, whereas the microscopic technique sampled approximately 250 cells. In order to compare the percent rosette obtained by each method, both Cytograf and microscopic rosette determinations were carried out on replicate l y m p h o c y t e suspensions from 27 donors (table 4). The mean % rosettes + standard deviation (S.D.) obtained by the t w o techniques for each donor were consistently similar (only 5 of the 27 pairs had significant differences). The rosette values obtained by each method are shown in fig. 3 (X axis, mi-
268 LINEAR PLOTOF CYTOGRAFVERSUS MICROSCOPIC DETERMINATION OF PERCENTROSETTE FORMING CELLS (RFC) FROM 27 VOLUNTEERS I0o
80
/// ///////
'~
e e
~ 4o ~:
20
2~o
4'0
o'o
B'o
,~o
% RFC (Microscopic Anolysis)
Fig. 3. Data from 27 volunteers comparing percent rosette forming cells (RFC) obtained by Cytograf 6300A versus microscopic analysis. Points have correlation coefficient (R) of 0.72 for dotted line (Y = 11.23 + 0.754X). Solid line represents theoretical 1 to 1 correlation of the two parameters. c r o s c o p i c analysis; Y axis, C y t o g r a f analysis). T h e d o t t e d line (Y = 11.23 + 0 . 7 5 4 X ) r e p r e s e n t s the line o f best fit f o r the 27 p o i n t s and has a c o r r e l a t i o n c o e f f i c i e n t o f 0.72 ( p e r f e c t c o r r e l a t i o n = 1.0) which indicates a highly significant degree o f c o r r e l a t i o n o f the t w o m e t h o d s (P < 0.01). DISCUSSION T h e p r o c e d u r e f o r q u a n t i t a t i n g T cells b y measuring sheep e r y t h r o c y t e rosettes is simple and specific, b u t the discrimination o f r o s e t t e s f r o m nonr o s e t t e d l y m p h o c y t e s o f t e n requires t e d i o u s m i c r o s c o p i c e x a m i n a t i o n o f a limited sampling o f cells. This r e p o r t describes the use o f the C y t o g r a f 6 3 0 0 A (Bio/Physics) t o a u t o m a t i c a l l y q u a n t i t a t e large n u m b e r s o f r o s e t t e - f o r m i n g cells. We have f o u n d t h a t this i n s t r u m e n t saves considerable t i m e and e f f o r t w h e n d e t e r m i n i n g R F C in multiple samples o n a r o u t i n e basis. The C y t o g r a f 6 3 0 0 A analysis samples a p p r o x . 100 times the n u m b e r o f cells and quantitates R F C r e p r o d u c i b l y with g o o d c o r r e l a t i o n t o m i c r o s c o p i c a l l y d e t e r m i n e d R F C percents. T h e d a t a in table 1 indicate t h a t a single c o n t r o l setting 09 = 34) can accur a t e l y a p p r o x i m a t e t h e p e r c e n t R F C for five representative individuals over a range o f m i c r o s c o p i c a l l y d e t e r m i n e d values (44--59%). T h e size distrib u t i o n o f l y m p h o c y t e s f r o m these individuals ( w h o include 2 c a n c e r p a t i e n t s and 3 n o r m a l v o l u n t e e r s ) shows considerable visual variation (on the oscilloscope); h o w e v e r , t h e p e r c e n t o f n o n - r o s e t t e d overlapping cells t h a t are inc l u d e d in the selected c o u n t (rosette) w i n d o w o n l y ranges f r o m 2.1--3.1%. It is likely t h a t a C y t o g r a f analysis o f R F C f r o m an individual with a signifi-
269 cantly smaller l y m p h o c y t e size distribution would be inaccurate, since many of the smaller rosettes might be deleted from the selected window (with a p = 34 setting). Indeed, one of our initial determinations showed only 0.7% overlapping cells, and correspondingly showed a large discrepancy in Cytograf vs microscopic RFC analysis (46 vs 62%). Individuals with less than 1.5% overlapping cells are currently being retested with the p control set to include an arbitrary 2.5% overlapping cells. Overlapping cell percents in the higher ranges (as high as 8.5%) did not result in significant differences in Cytograf and microscopic RFC values. The data presented in table 2 indicate that a two-fold increase in the number of cells counted above 35,000 results in an erroneous Cytograf determination of % RFC. This is probably the result of coincident particle counting, e.g. the Cytograf sensors detect two or more closely positioned smaller particles as a single large particle. The effects of coincidence increases the number of particles in the selected count window and thus increases the % RFC as observed. The Cytograf 6300A gives highly reproducible results when sequential replicate determinations are carried out first on the rosette preparation and then on the ZAP treated suspension. This data is included for practical reasons only. Replicate determinations performed on rosettes from several individuals is slowed by the thorough cleaning of the sample chamber that is required after each ZAP treated suspension is counted. This data indicates that the operator can complete the rosette count for all the samples to be tested (with minimal cleanings) and then count all the ZAP treated suspensions (with minimal intermittent cleaning). Rosette analysis performed using alternating rosetted and ZAP treated preparations (with thorough washing in between) allows statistical analysis of replicate determinations of the same sample which can be compared to replicate microscopic analysis of RFC% (table 3). This data indicates that the variance of the Cytograf analysis done in this manner is comparable to the variance in microscopic analysis performed by three experienced personnel. The two step Cytograf procedure outlined above allows quantitation of total lymphocytes, large lymphocytes, and RFC from the same tube. Attempts to measure total and large lymphocytes in a separate tube from the RFC (thus avoiding the lytic agent) have resulted in increased sample variation. Table 4 shows that the mean RFC percents obtained by Cytograf analysis of replicate tubes from 27 volunteers (including cancer patients and normal individuals) are very similar to microscopically determined values. The variation in RFC% from replicate tubes is reflected by the mean of the standard deviation, which is similar for the two methods (Cytograf, 3.9; microscope, 4.3). The plot of the data in table 4 (fig. 3) shows a highly significant correlation (P ~ 0.01) in % RFC determined by the two analyses (correlation coefficient R of the points for the line of best fit = 0.72). This correlation is represented by 22 of 27 samples t h a t are n o t different statistically, and 5 of 27 that are different. In one of the latter samples a predominantly small
270 l y m p h o c y t e p o p u l a t i o n biased the C y t o g r a f result (discussed above), while in the r e m a i n d e r , the d i s c r e p a n c y m i g h t be a t t r i b u t e d t o subjective errors associated with m i c r o s c o p i c evaluation. T h e C y t o g r a f t e c h n i q u e d e s c r i b e d above is easily a d a p t e d to d e t e r m i n e p e r c e n t o f live cells t h a t f o r m rosettes. T h e o n l y steps t h a t n e e d be a d d e d are, 1) t o s u p p l e m e n t the Z A P e d l y m p h o c y t e suspension with a vital d y e ( T r y p a n Blue), and 2) t o q u a n t i t a t e p e r c e n t viable l y m p h o c y t e s on the C y t o g r a f b y t h e m e t h o d o f Wilson et al. (1973). P e r c e n t R F C w o u l d t h a n be calculated b y using viable instead o f t o t a l l y m p h o c y t e s . ACKNOWLEDGEMENTS We are g r a t e f u l f o r the assistance o f H a t s u m i Park and Patricia Finke.
REFERENCES Bach, J.F., 1973, Transplant. Rev. 16, 196. Coombs, R.R.A., B.W. Gurner, A.B. Wilson, G. Holm and B. Lindgren, 1970, Int. Arch. Allergy 39,658. Fr¢land, S.S., 1972, Scand. J. Immunol. 1,269. Harris, R. and E.O. Ukaejiofo, 1969, Lancet 2,327. Jondal, M., G. Holm and H. Wigzell, 1972, J. Exp. Med. 136,207. Papamichail, M., E.J. Holborow, H.I. Keith et al., 1972, Lancet 2, 64. Silveira, N.P.A., N.F. Mendes and M.E.A. Tolnai, 1972, J. Immunol. 108, 1456. Wilson, M., J. Dardano and H. Rothschild, 1973, Transplantation 16,408. Wortis, H.H., A.G. Cooper and M.C. Brown, 1973, Nature New Biol. 243,109. Wybran, J., M.C. Carr and H.H. Fudenberg, 1972, J. Clin. Invest. 51, 2537.