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The mixed lymphocyte response in whole blood: technical aspects
Journal of Immunological Methods, 21 (1978) 237--245 © Elsevier/North-Holland Biomedical Press
237
THE MIXED LYMPHOCYTE RESPONSE IN WHOLE BLOOD: TECHNICAL ASPECTS
W. RELLA Institute for Cancer Research, University of Vienna, A-1090 Vienna, Austria
(Received 9 December 1977, accepted 10 January 1978)
Experiments were conducted to standardize the response of lymphocytes in whole blood in mixed culture with allogeneic lymphocytes. The following conditio~is were found suitable: (1) A culture period of 6--8 days. (2) The ratio of stimulator to responder lymphocytes should be 4--8, but may vary from one batch of stimulator cells to another. (3) Tests may be performed in culture tubes with 50--100 pl blood in a total volume of 2 ml (macrotechnique) or in microplates with 10 pl of blood in a volume of 0.2 ml (microtechnique). (4) Serum supplement is not required. (5) Results should be expressed as counts/min (cpm)per a given number of responder lymphocytes. Stimulation indices are less reliable.
INTRODUCTION F u n c t i o n a l tests f o r h u m a n l y m p h o c y t e s have b e c o m e r o u t i n e in m a n y clinical i m m u n o l o g y l a b o r a t o r i e s . R e c e n t l y , w h o l e - b l o o d c u l t u r e s have b e e n i n t r o d u c e d as an a l t e r n a t i v e t o c o n v e n t i o n a l m e t h o d s using isolated l y m p h o c y t e s ( P a t y and Hughes, 1 9 7 2 ; S t o c k m a n et al., 1 9 7 2 ; Pellegrino et al., 1 9 7 3 ) . It has b e e n s h o w n t h a t t h e p r e s e n c e o f o t h e r b l o o d e l e m e n t s d o e s n o t a f f e c t t h e s t i m u l a t o r y r e s p o n s e o f t h e l y m p h o c y t e s . W h o l e - b l o o d c u l t u r e systems offer the advantages of minimal blood requirement, technical simplicity, a n d m i n i m a l a l t e r a t i o n o f cell e l e m e n t s b y m a n i p u l a t i o n a n d i s o l a t i o n p r o c e dures. O n e m a j o r d i s a d v a n t a g e , h o w e v e r , is t h e variable c o n t e n t o f l y m p h o c y t e s in such c u l t u r e s d u e t o daily f l u c t u a t i o n s in t h e d o n o r b l o o d . This i n t r o d u c e s an e l e m e n t o f u n c e r t a i n t y w h i c h m a y have d i s c o u r a g e d m o r e general use o f w h o l e - b l o o d cultures. T h e p r e s e n t p a p e r investigates t h e influence o f d i f f e r i n g n u m b e r s o f r e s p o n d e r l y m p h o c y t e s in c u l t u r e a n d o f f e r s a possibility o f p a r t i a l l y c i r c u m v e n t i n g this p r o b l e m . In a d d i t i o n , t h e a d a p t a t i o n o f w h o l e - b l o o d c u l t u r e s t o m i c r o p l a t e s is described.
238 MATERIALS AND METHODS
Blood samples and lymphocyte counts Venous blood from healthy donors and sick children was drawn in a plastic syringe prerinsed with Liquemin ®. Total leukocytes were c o u n t e d in Tiirk's solution using a h e m o c y t o m e t e r and the percentage of l y m p h o c y t e s was determined per 100 nucleated cells stained with May--Griinwald-Giemsa.
Isolation of lymphocytes Mononuclear l y m p h o i d cells were separated from whole blood by density flotation on Ficoll--Hypaque (Perper et al., 1968).
Stimulator cells Stimulator cells were peripheral blood l y m p h o c y t e s isolated from the b u f f y coat o f blood samples from professional donors. The l y m p h o c y t e s were irradiated with 6000 R from a 6°Co source (courtesy of Dr. Binder, Dept. of Radiotherapy) and were frozen in 2 ml aliquots (10 ~ cells/ml) in liquid N2. Freezing and thawing procedures were p e r f o r m e d essentially as described by Strong and Sell (1977).
Blood cultures Macrotechnique. While the defrosted stimulator l y m p h o c y t e s were being washed, cultures of the responding blood were prepared by mixing 1 vol of whole heparinized blood with 9 vol of m edi um RPMI 1640 (GIBCO) supplem en ted with 2 mM glutamine and antibiotics. Of the diluted blood 1 ml was pipetted into a series of disposable tubes (17 mm × 100 mm, Falcon 2001). Immediately, an optimal n u m b e r o f irradiated stimulator l y m p h o c y t e s suspended in 1 ml of m e di um were added and the cultures were put in the CO2 incubator (37°C, 8% CO2 in water-saturated air). The cultures were pulsed with 1 pCi (0.1 ml) of [3H]thymidine before the last 14--16 h of incubation. The cultured cells were then lysed with distilled water and particulate material was collected on glass fiber paper with a multiharvester device, and dried. The radioactivity retained on the filter was furt her processed for liquid scintillation counting. Results were recorded as count s/ m i n (cpm); the counting efficiency was 67%. Microtechnique. Microtiter plates II, with 8 × 12 r o u n d - b o t t o m e d wells (NUNC), were used. Of heparinized blood 1 vol was diluted with 19 vol of medium and 0.2 ml of the suspension placed in each well. Irradiated stimulator cells (prepared as above) were suspended in an appropriate volume and 20 pl added to each well f r om a disposable 1 ml syringe fitted to a Hamilton dispenser. Cultures were set up in quadruplicate with and w i t h o u t stimulator cells. 0.2 gCi (20 pl) [3H]thymidine were added 14--16 h before the end of the culture period, and the cultures were processed as described above.
239 RESULTS
(1) Kinetic o f the mixed lymphocyte culture (MLC) 100 pl o f whole blood were co-cultured with an optimal n u m b e r of stimulator cells in 2 ml culture volume for 5, 6, 7, 8 or 9 days. The results are given in Fig. 1. Maximal isotope uptake occurs on day 8, but good discriminatory results are obtained also on day 6 or 7. Routinely, day 7 was chosen as the e n d p o i n t of culture.
(2) Variation o f volume o f diluted blood and serum supplement The volume of blood had a considerable influence on MLC stimulation. Isotope uptake per cultured l y m p h o c y t e increased linearly with volume (Fig. 2). As a routine, 100 pl of whole diluted blood were chosen for each macroculture and 10 #1 f or microcultures, although o t h e r volumes might do as well. Supplementing cultures with either human AB/serum or fetal bovine serum (5--15%) had a variable effect upon MLC stimulation, but the net result was f r e q u e n t l y worse with serum than w i t hout serum. In order not to introduce a new source of variation, cultures were set up routinely w i t h o u t serum supplement.
(3) Influence o f the number o f stimulating cells Fig. 3 shows 5 examples in which different numbers of irradiated lymphocytes were used to stimulate a constant volume of blood. The optimal
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Fig. 1. Kinetic o f t h e M L C r e s p o n s e w i t h w h o l e b l o o d ( m a c r o t e c h n i q u e ) . Vertical bars i n d i c a t e t h e S.D. o f t r i p l i c a t e cultures. Fig. 2. E f f e c t o f v o l u m e o f d i l u t e d b l o o d o n t h e MLC r e s p o n s e ( m a c r o c u l t u r e ) . I n d i c a t e d v o l u m e s of b l o o d were c u l t u r e d in 2 ml m e d i u m w i t h a c o n s t a n t n u m b e r o f s t i m u l a t o r cells for 7 days. C p m p e r 10 s l y m p h o c y t e s (+- S.D.) are s h o w n .
240 60.
7
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Stimulotor/Resporx~r Ratio
Fig. 3. I n f l u e n c e o f d i f f e r e n t s t i m u l a t o r t o r e s p o n d e r cell ratios in the w h o l e b l o o d MLC. G r a d e d n u m b e r s o f s t i m u l a t o r cells were a d d e d t o a c o n s t a n t v o l u m e o f b l o o d .
stimulator--responder ratio varied with different batches of stimulator lymphocytes. Stimulator--responder ratios of 4 : 1--8 : 1 were usually satisfactory. However, it may be advisable to study the dose--response relationship for each new batch of irradiated lymphocytes.
(4) Comparison of macro- and microcultures Blood samples from 22 children with presumed defects in cell-mediated immunity were assayed by macro- and microtechniques performed in parallel.
200
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100
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E Q.
Fig. 4. C o m p a r i s o n o f macro- and m i c r o - w h o l e - b l o o d MLC. C o l u m n s r e p r e s e n t c p m of m a c r o c u l t u r e s ; vertical bars w i t h i n c o l u m n s r e p r e s e n t c p m o f parallel m i c r o c u l t u r e s x 10.
241
For macrocultures, 100 t~l of blood were cultured in 2 ml medium and were pulsed with 1 pCi of [3H]thymidine. For microcultures, 10 t~l of blood were cultured in 0.2 ml of medium and were pulsed with 0.2 pCi of [3H]thymidine. The stimulator--responder cell ratio and the length of culture period were equivalent. The results are shown in Fig. 4. Cpm of microcultures were multiplied by 10 to allow comparison with macrocultures. Thymidine uptake was usually higher in microcultures due to the higher concentration of [3H]thymidine in the medium. The correlation coefficient for the two techniques was 0.80 when cpm were compared, b u t only 0.57 when stimulation indices (not shown) were compared. Both methods agreed in placing the 4 children at the right as low responders.
(5) Effect of the number of responder lymphocytes The influence of the number of responder lymphocytes on the MLC response was studied as follows: (a) By noting the MLC response of various healthy volunteers with differing l y m p h o c y t e counts in the peripheral blood (Fig. 5). 1607
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Fig. 5. W h o l e - b l o o d M L C r e s p o n s e o f 10 d i f f e r e n t h e a l t h y d o n o r s ( m a c r o t e c h n i q u e ) . Results are e x p r e s s e d as n e t c p m ( t o p ) , as s t i m u l a t i o n i n d e x ( c p m r a t i o of s t i m u l a t e d / u n s t i m u l a t e d c u l t u r e s , m i d d l e ) or c p m / 1 0 s l y m p h o c y t e s ( b o t t o m ) .
242
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Fig. 6. MLC response of whole blood (microtechnique), depleted of, or enriched with, lymphocytes. The number of stimulator cells was kept constant, o, total uptake; o, uptake/lymphocyte.
(b) By measuring the sequential responses in 2 donors over a time period during which the l y m p h o c y t e c o u n t varied (Table 1). (c) By measuring MLC responses in one blood sample in one test in which l y m p h o c y t e s were artificially enriched or depleted (Fig. 6). Fig. 5 clearly shows t ha t the variation in the MLC response between healthy donors is least if results are expressed as cpm per l y m p h o c y t e (number 10, apparently, shows partial LD identity). Table 1 points to the same conclusion. Over a period o f ' 8 weeks stimulation indices of a given d o n o r stimulated with the same batch of stimulator cells varied 3--8-fold while c p m / l y m p h o c y t e varied only 1.5-fold. In Fig. 6 whole blood was first depleted o f l y m p h o c y t e s by density centrifugation. Graded numbers of washed l y m p h o c y t e s were t hen re-added again and each suspension was tested in the MLC. Data f r om 3 experiments are shown in the figure. The total isotope uptake in the culture increases as more l y m p h o c y t e s are added and reaches a m a x i m u m at about 4000 l y m p h o c y t e s / m m 3. Isotope uptake per l y m p h o c y t e , however, remains relatively constant between 700 up to a b o u t 4000 l y m p h o c y t e s / m m 3 and t he n decreases. DISCUSSION The whole-blood MLC, as shown here, can be rendered relatively resistant to the effects of intrinsic variations in the test system, provided certain requirements are fulfilled. The l y m p h o c y t e c o u n t in whole blood appears n o t to be a major cause o f variation, if results are expressed as c p m / l y m p h o cyte. On the o ther hand, stimulation indices are not so reliable, due to un-
244 predictable isotope uptake in unstimulated cultures. Blood samples with more than 4000 l y m p h o c y t e s / m m 3 showed lower incorporation of thymidine per lymphocyte. Similar observations have been reported for mitogen stimulation by Hall and Gordon (1976), although they gave no details on this point. Since high l y m p h o c y t e counts are normally seen in young children, it may be better when their blood is being tested to use half the volume. The volume of blood, once chosen, should be kept constant throughout a given series of tests, since, as shown in Fig. 2, the volume influences the range of reactivity. High stimulator--responder ratios were required to obtain optimal stimulation in the whole-blood MLC. This may be because the probability of lymphocyte interaction leading to activation is less in whole blood, where many erythrocytes are interspersed. It appears advisable to test the range of optimal stimulation for each new batch of lymphocytes, although standardization of l y m p h o c y t e subpopulations amongst stimulator cells might make this requirement unnecessary. Comparison of macro- and microcultures shows that MLC tests can be carried out with as few as 10 pl blood per culture. Rapid and accurate distribution of cells and reagents into the wells is achieved by the use of a disposable syringe attached to the repeating dispenser. Mixing of added material is not necessary with this technique since the jet of medium released by the dispenser mixes the culture. Eskola et al. (1975) and Viljanen and Eskola ( 1 9 7 7 ) h a v e introduced a similar microtechnique for mitogens or PPD, using 25--50 ~l of blood. In our culture system, the efficient use of only 10 pl of blood for the MLC, as well as for mitogen or antigen stimulation (unpublished), offers, for the first time, the possibility of studying the stimulatory properties of capillary blood. Preliminary experiments using blood from the fingertip have given encouraging results. Given appropriate conditions, whole-blood cultures can be recommended as routine tests for follow-up functional activity of lymphocytes. In certain circumstances, whole blood may also offer an alternative method of testing for HLA-D antigens. Nevertheless, whole-blood cultures remain crude tests in which the relative role of l y m p h o c y t e subpopulations and possible influences of autologous plasma factors are mostly unknown. ACKNOWLEDGEMENTS I wish to t h a n k those who donated their blood for this study. The technical assistance of Miss Elke Fritz and Mrs. Leopoldine Oedendorfer is gratefully acknowledged. REFERENCES
Eskola, J., E. Soppi, M. Viljanen and O. Ruuskanen, 1975, Immunol. Comm. 4, 297. Hall, L.S. and D.S. Gordon, 1976, J. Immunol. Methods 12, 31.
245 Paty, D.W. and D. Hughes, 1972, J. Immunol. Methods 2, 99. Pellegrino, M.A., S. Ferone, A. Pellegrino and R.A. Reisfeld, 1973, Clin. Immunol. Immunopathol. 2, 67. Perper, R.J., T.W. Zee and M.M. Mickelson, 1968, J. Lab. Clin. Med. 72, 842. Stockman, G.D., D.M. Mumford and J.R. Wilbur, 1972, Int. Arch. Allergy 43, 759. Strong, D.M. and K.W. Sell, 1977, Colloque Cryoimmunologie 1976 (INSERM, Paris) 81 pp. Vilkanen, M.K. and 3. Eskola, 1977, Clin. Immunol. Immunopathol. 8, 28.
237
THE MIXED LYMPHOCYTE RESPONSE IN WHOLE BLOOD: TECHNICAL ASPECTS
W. RELLA Institute for Cancer Research, University of Vienna, A-1090 Vienna, Austria
(Received 9 December 1977, accepted 10 January 1978)
Experiments were conducted to standardize the response of lymphocytes in whole blood in mixed culture with allogeneic lymphocytes. The following conditio~is were found suitable: (1) A culture period of 6--8 days. (2) The ratio of stimulator to responder lymphocytes should be 4--8, but may vary from one batch of stimulator cells to another. (3) Tests may be performed in culture tubes with 50--100 pl blood in a total volume of 2 ml (macrotechnique) or in microplates with 10 pl of blood in a volume of 0.2 ml (microtechnique). (4) Serum supplement is not required. (5) Results should be expressed as counts/min (cpm)per a given number of responder lymphocytes. Stimulation indices are less reliable.
INTRODUCTION F u n c t i o n a l tests f o r h u m a n l y m p h o c y t e s have b e c o m e r o u t i n e in m a n y clinical i m m u n o l o g y l a b o r a t o r i e s . R e c e n t l y , w h o l e - b l o o d c u l t u r e s have b e e n i n t r o d u c e d as an a l t e r n a t i v e t o c o n v e n t i o n a l m e t h o d s using isolated l y m p h o c y t e s ( P a t y and Hughes, 1 9 7 2 ; S t o c k m a n et al., 1 9 7 2 ; Pellegrino et al., 1 9 7 3 ) . It has b e e n s h o w n t h a t t h e p r e s e n c e o f o t h e r b l o o d e l e m e n t s d o e s n o t a f f e c t t h e s t i m u l a t o r y r e s p o n s e o f t h e l y m p h o c y t e s . W h o l e - b l o o d c u l t u r e systems offer the advantages of minimal blood requirement, technical simplicity, a n d m i n i m a l a l t e r a t i o n o f cell e l e m e n t s b y m a n i p u l a t i o n a n d i s o l a t i o n p r o c e dures. O n e m a j o r d i s a d v a n t a g e , h o w e v e r , is t h e variable c o n t e n t o f l y m p h o c y t e s in such c u l t u r e s d u e t o daily f l u c t u a t i o n s in t h e d o n o r b l o o d . This i n t r o d u c e s an e l e m e n t o f u n c e r t a i n t y w h i c h m a y have d i s c o u r a g e d m o r e general use o f w h o l e - b l o o d cultures. T h e p r e s e n t p a p e r investigates t h e influence o f d i f f e r i n g n u m b e r s o f r e s p o n d e r l y m p h o c y t e s in c u l t u r e a n d o f f e r s a possibility o f p a r t i a l l y c i r c u m v e n t i n g this p r o b l e m . In a d d i t i o n , t h e a d a p t a t i o n o f w h o l e - b l o o d c u l t u r e s t o m i c r o p l a t e s is described.
238 MATERIALS AND METHODS
Blood samples and lymphocyte counts Venous blood from healthy donors and sick children was drawn in a plastic syringe prerinsed with Liquemin ®. Total leukocytes were c o u n t e d in Tiirk's solution using a h e m o c y t o m e t e r and the percentage of l y m p h o c y t e s was determined per 100 nucleated cells stained with May--Griinwald-Giemsa.
Isolation of lymphocytes Mononuclear l y m p h o i d cells were separated from whole blood by density flotation on Ficoll--Hypaque (Perper et al., 1968).
Stimulator cells Stimulator cells were peripheral blood l y m p h o c y t e s isolated from the b u f f y coat o f blood samples from professional donors. The l y m p h o c y t e s were irradiated with 6000 R from a 6°Co source (courtesy of Dr. Binder, Dept. of Radiotherapy) and were frozen in 2 ml aliquots (10 ~ cells/ml) in liquid N2. Freezing and thawing procedures were p e r f o r m e d essentially as described by Strong and Sell (1977).
Blood cultures Macrotechnique. While the defrosted stimulator l y m p h o c y t e s were being washed, cultures of the responding blood were prepared by mixing 1 vol of whole heparinized blood with 9 vol of m edi um RPMI 1640 (GIBCO) supplem en ted with 2 mM glutamine and antibiotics. Of the diluted blood 1 ml was pipetted into a series of disposable tubes (17 mm × 100 mm, Falcon 2001). Immediately, an optimal n u m b e r o f irradiated stimulator l y m p h o c y t e s suspended in 1 ml of m e di um were added and the cultures were put in the CO2 incubator (37°C, 8% CO2 in water-saturated air). The cultures were pulsed with 1 pCi (0.1 ml) of [3H]thymidine before the last 14--16 h of incubation. The cultured cells were then lysed with distilled water and particulate material was collected on glass fiber paper with a multiharvester device, and dried. The radioactivity retained on the filter was furt her processed for liquid scintillation counting. Results were recorded as count s/ m i n (cpm); the counting efficiency was 67%. Microtechnique. Microtiter plates II, with 8 × 12 r o u n d - b o t t o m e d wells (NUNC), were used. Of heparinized blood 1 vol was diluted with 19 vol of medium and 0.2 ml of the suspension placed in each well. Irradiated stimulator cells (prepared as above) were suspended in an appropriate volume and 20 pl added to each well f r om a disposable 1 ml syringe fitted to a Hamilton dispenser. Cultures were set up in quadruplicate with and w i t h o u t stimulator cells. 0.2 gCi (20 pl) [3H]thymidine were added 14--16 h before the end of the culture period, and the cultures were processed as described above.
239 RESULTS
(1) Kinetic o f the mixed lymphocyte culture (MLC) 100 pl o f whole blood were co-cultured with an optimal n u m b e r of stimulator cells in 2 ml culture volume for 5, 6, 7, 8 or 9 days. The results are given in Fig. 1. Maximal isotope uptake occurs on day 8, but good discriminatory results are obtained also on day 6 or 7. Routinely, day 7 was chosen as the e n d p o i n t of culture.
(2) Variation o f volume o f diluted blood and serum supplement The volume of blood had a considerable influence on MLC stimulation. Isotope uptake per cultured l y m p h o c y t e increased linearly with volume (Fig. 2). As a routine, 100 pl of whole diluted blood were chosen for each macroculture and 10 #1 f or microcultures, although o t h e r volumes might do as well. Supplementing cultures with either human AB/serum or fetal bovine serum (5--15%) had a variable effect upon MLC stimulation, but the net result was f r e q u e n t l y worse with serum than w i t hout serum. In order not to introduce a new source of variation, cultures were set up routinely w i t h o u t serum supplement.
(3) Influence o f the number o f stimulating cells Fig. 3 shows 5 examples in which different numbers of irradiated lymphocytes were used to stimulate a constant volume of blood. The optimal
~ 60x ~
100 ¸
50-
80
x
E
o
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2O
TO-
; 5
"~ 6
7 7
7 8
[ ~ y s in Culture
". 9
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Whole Blood
Fig. 1. Kinetic o f t h e M L C r e s p o n s e w i t h w h o l e b l o o d ( m a c r o t e c h n i q u e ) . Vertical bars i n d i c a t e t h e S.D. o f t r i p l i c a t e cultures. Fig. 2. E f f e c t o f v o l u m e o f d i l u t e d b l o o d o n t h e MLC r e s p o n s e ( m a c r o c u l t u r e ) . I n d i c a t e d v o l u m e s of b l o o d were c u l t u r e d in 2 ml m e d i u m w i t h a c o n s t a n t n u m b e r o f s t i m u l a t o r cells for 7 days. C p m p e r 10 s l y m p h o c y t e s (+- S.D.) are s h o w n .
240 60.
7
E
Stimulotor/Resporx~r Ratio
Fig. 3. I n f l u e n c e o f d i f f e r e n t s t i m u l a t o r t o r e s p o n d e r cell ratios in the w h o l e b l o o d MLC. G r a d e d n u m b e r s o f s t i m u l a t o r cells were a d d e d t o a c o n s t a n t v o l u m e o f b l o o d .
stimulator--responder ratio varied with different batches of stimulator lymphocytes. Stimulator--responder ratios of 4 : 1--8 : 1 were usually satisfactory. However, it may be advisable to study the dose--response relationship for each new batch of irradiated lymphocytes.
(4) Comparison of macro- and microcultures Blood samples from 22 children with presumed defects in cell-mediated immunity were assayed by macro- and microtechniques performed in parallel.
200
I
%
100
X
E Q.
Fig. 4. C o m p a r i s o n o f macro- and m i c r o - w h o l e - b l o o d MLC. C o l u m n s r e p r e s e n t c p m of m a c r o c u l t u r e s ; vertical bars w i t h i n c o l u m n s r e p r e s e n t c p m o f parallel m i c r o c u l t u r e s x 10.
241
For macrocultures, 100 t~l of blood were cultured in 2 ml medium and were pulsed with 1 pCi of [3H]thymidine. For microcultures, 10 t~l of blood were cultured in 0.2 ml of medium and were pulsed with 0.2 pCi of [3H]thymidine. The stimulator--responder cell ratio and the length of culture period were equivalent. The results are shown in Fig. 4. Cpm of microcultures were multiplied by 10 to allow comparison with macrocultures. Thymidine uptake was usually higher in microcultures due to the higher concentration of [3H]thymidine in the medium. The correlation coefficient for the two techniques was 0.80 when cpm were compared, b u t only 0.57 when stimulation indices (not shown) were compared. Both methods agreed in placing the 4 children at the right as low responders.
(5) Effect of the number of responder lymphocytes The influence of the number of responder lymphocytes on the MLC response was studied as follows: (a) By noting the MLC response of various healthy volunteers with differing l y m p h o c y t e counts in the peripheral blood (Fig. 5). 1607
120
x
0
200 1.543
.Eso~
40. 30" ×
_~2o-
234 5678910
Fig. 5. W h o l e - b l o o d M L C r e s p o n s e o f 10 d i f f e r e n t h e a l t h y d o n o r s ( m a c r o t e c h n i q u e ) . Results are e x p r e s s e d as n e t c p m ( t o p ) , as s t i m u l a t i o n i n d e x ( c p m r a t i o of s t i m u l a t e d / u n s t i m u l a t e d c u l t u r e s , m i d d l e ) or c p m / 1 0 s l y m p h o c y t e s ( b o t t o m ) .
242
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Fig. 6. MLC response of whole blood (microtechnique), depleted of, or enriched with, lymphocytes. The number of stimulator cells was kept constant, o, total uptake; o, uptake/lymphocyte.
(b) By measuring the sequential responses in 2 donors over a time period during which the l y m p h o c y t e c o u n t varied (Table 1). (c) By measuring MLC responses in one blood sample in one test in which l y m p h o c y t e s were artificially enriched or depleted (Fig. 6). Fig. 5 clearly shows t ha t the variation in the MLC response between healthy donors is least if results are expressed as cpm per l y m p h o c y t e (number 10, apparently, shows partial LD identity). Table 1 points to the same conclusion. Over a period o f ' 8 weeks stimulation indices of a given d o n o r stimulated with the same batch of stimulator cells varied 3--8-fold while c p m / l y m p h o c y t e varied only 1.5-fold. In Fig. 6 whole blood was first depleted o f l y m p h o c y t e s by density centrifugation. Graded numbers of washed l y m p h o c y t e s were t hen re-added again and each suspension was tested in the MLC. Data f r om 3 experiments are shown in the figure. The total isotope uptake in the culture increases as more l y m p h o c y t e s are added and reaches a m a x i m u m at about 4000 l y m p h o c y t e s / m m 3. Isotope uptake per l y m p h o c y t e , however, remains relatively constant between 700 up to a b o u t 4000 l y m p h o c y t e s / m m 3 and t he n decreases. DISCUSSION The whole-blood MLC, as shown here, can be rendered relatively resistant to the effects of intrinsic variations in the test system, provided certain requirements are fulfilled. The l y m p h o c y t e c o u n t in whole blood appears n o t to be a major cause o f variation, if results are expressed as c p m / l y m p h o cyte. On the o ther hand, stimulation indices are not so reliable, due to un-
244 predictable isotope uptake in unstimulated cultures. Blood samples with more than 4000 l y m p h o c y t e s / m m 3 showed lower incorporation of thymidine per lymphocyte. Similar observations have been reported for mitogen stimulation by Hall and Gordon (1976), although they gave no details on this point. Since high l y m p h o c y t e counts are normally seen in young children, it may be better when their blood is being tested to use half the volume. The volume of blood, once chosen, should be kept constant throughout a given series of tests, since, as shown in Fig. 2, the volume influences the range of reactivity. High stimulator--responder ratios were required to obtain optimal stimulation in the whole-blood MLC. This may be because the probability of lymphocyte interaction leading to activation is less in whole blood, where many erythrocytes are interspersed. It appears advisable to test the range of optimal stimulation for each new batch of lymphocytes, although standardization of l y m p h o c y t e subpopulations amongst stimulator cells might make this requirement unnecessary. Comparison of macro- and microcultures shows that MLC tests can be carried out with as few as 10 pl blood per culture. Rapid and accurate distribution of cells and reagents into the wells is achieved by the use of a disposable syringe attached to the repeating dispenser. Mixing of added material is not necessary with this technique since the jet of medium released by the dispenser mixes the culture. Eskola et al. (1975) and Viljanen and Eskola ( 1 9 7 7 ) h a v e introduced a similar microtechnique for mitogens or PPD, using 25--50 ~l of blood. In our culture system, the efficient use of only 10 pl of blood for the MLC, as well as for mitogen or antigen stimulation (unpublished), offers, for the first time, the possibility of studying the stimulatory properties of capillary blood. Preliminary experiments using blood from the fingertip have given encouraging results. Given appropriate conditions, whole-blood cultures can be recommended as routine tests for follow-up functional activity of lymphocytes. In certain circumstances, whole blood may also offer an alternative method of testing for HLA-D antigens. Nevertheless, whole-blood cultures remain crude tests in which the relative role of l y m p h o c y t e subpopulations and possible influences of autologous plasma factors are mostly unknown. ACKNOWLEDGEMENTS I wish to t h a n k those who donated their blood for this study. The technical assistance of Miss Elke Fritz and Mrs. Leopoldine Oedendorfer is gratefully acknowledged. REFERENCES
Eskola, J., E. Soppi, M. Viljanen and O. Ruuskanen, 1975, Immunol. Comm. 4, 297. Hall, L.S. and D.S. Gordon, 1976, J. Immunol. Methods 12, 31.
245 Paty, D.W. and D. Hughes, 1972, J. Immunol. Methods 2, 99. Pellegrino, M.A., S. Ferone, A. Pellegrino and R.A. Reisfeld, 1973, Clin. Immunol. Immunopathol. 2, 67. Perper, R.J., T.W. Zee and M.M. Mickelson, 1968, J. Lab. Clin. Med. 72, 842. Stockman, G.D., D.M. Mumford and J.R. Wilbur, 1972, Int. Arch. Allergy 43, 759. Strong, D.M. and K.W. Sell, 1977, Colloque Cryoimmunologie 1976 (INSERM, Paris) 81 pp. Vilkanen, M.K. and 3. Eskola, 1977, Clin. Immunol. Immunopathol. 8, 28.