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Mononuclear cells migration inhibition and delayed hypersensitivity in man
Journal of Immunological Methods, 11 ( 1 9 7 6 ) 1--6
1
© N o r t h - H o l l a n d P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
M O N O N U C L E A R CELLS MIGRATION INHIBITION AND D E L A Y E D HYPERSENSITIVITY IN MAN
R O N A L D P. NG a n d C O N S T A N T I ' N G. A L E X O P O U L O S
Department of Clinical Haematology, University College Hospital Medical School, London W.C.I., England (Received 9 S e p t e m b e r 1 9 7 5 , a c c e p t e d 9 N o v e m b e r 1 9 7 5 )
T h e m i g r a t i o n i n h i b i t i o n b y soluble PPD o f p e r i p h e r a l b l o o d m o n o n u c l e a r cells s h o w e d a m u c h b e t t e r r e f l e c t i o n of delayed h y p e r s e n s i t i v i t y t o t u b e r c u l i n in m a n t h a n t h e m i g r a t i o n i n h i b i t i o n of b u f f y c o a t cells. T h e m i g r a t i o n i n d e x (MI) w i t h m o n o n u c l e a r cells in a g r o u p of t u b e r c u l i n positive s u b j e c t s was 0.51 -+ 0.14 ( m e a n ± S.D.) (n = 20) and t h a t of a g r o u p of t u b e r c u l i n negative s u b j e c t s was 0.89 ± 0.11 (n = 14) (P < 0.001). Using b u f f y c o a t cells t h e MI was 0.65 -+ 0.26 (n = 19) for a g r o u p o f t u b e r c u l i n positive s u b j e c t s a n d 0 . 7 6 ± 0.24 (n = 10) for t h e g r o u p of t u b e r c u l i n negative subjects. T h e y were n o t significantly d i f f e r e n t (P > 0.05). T h e m o n o n u c l e a r cells m i g r a t i o n i n h i b i t i o n was also q u i t e r e p r o d u c i b l e f r o m day to day.
The Macrophage Migration Inhibition test is an accepted in vitro reflection of delayed hypersensitivity in guinea pigs (David et al., 1964; Bloom and Bennet, 1968). A similar technique using human l y m p h o c y t e culture supernatant and guinea pig peritoneal macrophages has also been developed {Thor et al., 1968; Rocklin et al., 1970). Although this indirect m e t h o d showed a good correlation with in vivo tests for delayed hypersensitivity, it could not be widely used in clinical situations because of its technical complexity. However, an in vitro m e t h o d (SOborg and Bendixen, 1967) based on inhibition of the migration of human peripheral blood b u f f y coat cells in the presence of Brucella antigen has been found to correlate well with skin test results (SCborg, 1967). A review of the literature since then shows that there are a number of m e t h o d s for doing such an in vitro test, using soluble or particulate antigens. When soluble antigens were used some investigators failed to show any correlation between the results of the test and delayed hypersensitivity in man (Kaltreider et al., 1969; Lockshin, 1969), while others could do so only with considerable effort and care (Rosenberg et al., 1970; Federlin et al., 1971; Mitchell et al., 1972). Several workers have suggested that only particulate antigens could given reliable results (Federlin et al., 1971; Gorski, 1974). Most authors measured the entire area of migration covered b y the cells; however Rosenberg and David (1974) found that only the inner dense area of migration showed inhibition by specific antigen in sensitive individuals, while the 'halo' of surrounding cells did n o t show in-
hibition of migration. They pointed out that about 90% of the cells in the 'halo' were polymorphs and only a few mononuclears were present. SOborg and Bendixen (1967) also found that the cells involved in the leukocytes Migration Inhibition Test were mononuclear cells. Because of our interest in clinical applications of the test, and in view of the discrepancies reported in the literature, we have investigated the relationship between the Leukocyte Migration Inhibition Test and delayed hypersensitivity in man using a soluble purified protein derivative (PPD) as the antigen together with pure preparations of mononuclear cells, and compared the results obtained with those using buffy coat preparations containing both mononuclear cells and polymorphs. MATERIALS AND METHODS
Subjects Thirty-two healthy adult volunteers, whose reaction to 10 units of tuberculin (T.U.) had recently been tested, were selected for study. In ten instances, blood from the umbilical cord of new born infants {assumed to be tuberculin negative), was used.
Preparation of cells Bully coat cells. Ten ml of blood was taken into heparinised plastic tubes and allowed to stand for 1 h at 37°C. The leukocyte rich plasma was then taken off, and centrifuged and the cells obtained were washed three times in phosphate buffered saline at pH 7.3. Mononuclear cells. Ten ml of blood was mixed with an equal volume of phosphate buffered saline and mononuclear cells (95% purity) were then obtained by centrifugation in a gradient of Ficoll--Triosil (Boyum, 1968). The cells were then washed three times in phosphate buffered saline. Migration test The preparations of buffy coat cells or mononuclear cells were taken into mmrohaematocrit tubes (internal diameter of 1 mm, and 75 m m in length) wi~ich were sealed with 'Cristaseal' and centrifuged (12,000 g) for 5 min. The tubes were cut at the interface between the fluid and the cell layer and secured into wells on plastic micro-migration plates (Sterilin). The wells were filled with a solution containing RPMI 1640 medium (Biocult) 9 vol, fetal calf serum 1 vol, Gentamycin 4 gg/ml and preservative-free PPD (Weybridge Central Veterinary Laboratory, Ministry of Agriculture) 100 pg/ml; in the control well PPD was omitted. The wells were sealed with glass cover-slips and silicone grease and placed horizontally into an incubator for 18 h at 37°C. The area of migration was projected and traced onto paper and the rel-
ative size of the projected areas measured by cutting out and weighing. All tests were carried out in triplicate. The coefficient of variation between replicates for the mononuclear cells migration inhibition test was 9.9% and that for the mixed leukocyte migration inhibition test was 18.2%. The migration areas in the presence and absence of antigen were compared and expressed as an index using the formula: Area of migration in presence of antigen Migration Index (MI) = Area of migration in absence of antigen In 15 subjects simultaneous migration tests were set up with both mononuclear cells and buffy coat cells. RESULTS AND DISCUSSION
Using mononuclear cells, the MI for a group of tuberculin positive subjects was 0.51 + 0.14 (mean + S.D.) (n = 20) and that for the tuberculin negative subjects was 0.89 + 0.11 (n = 14) and they are significantly different ( P < 0.001). The MI value of 0.73 was then chosen as the dividing point for separating the 2 groups, as 0.73 is 1.52 S.D. away from the mean of both groups.
b
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&&
h
•
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X w
.E
A & A
_'2
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0
:e .e e:e
Z .( Z 0
0
00 oo
.4--
o •
•
0
.2D
0
•
p pD +re
•
PPD -ve
• •
PPD PPD
+re -ve
Fig. 1. Migration Indices of tuberculin positive and tuberculin negative subjects using preparations of mononuclear cells. Fig. 2. Migration Indices of tuberculin positive and tuberculin negative subjects using preparations of buffy coat cells.
1.0
I.O
:E × w c~ Z
× w z
z 0
Z 0.5
..5
1 50
I 1OO
I 150
I
I 200
50
PPD pg/ml
I
IOO
I
I
150
200
PPD pg,'nq
Fig. 3. Migration Indices of 5 tuberculin positive subjects using preparations of buffy coat cells at different concentrations of PPD. Fig. 4. Migration Indices of 5 tuberculin positive subjects using mononuclear cells at different concentrations of PPD.
As can be seen f r o m fig. 1, there w a s n o overlap b e t w e e n t h e groups o f subjects studied. On a t h e o r e t i c a l basis a standard score (or z score) o f + 1 . 5 2 inc l u d e s 87% o f t h e p o p u l a t i o n and since a n y value that is less than m e a n - - 1 . 5 2 S.D. f r o m a t u b e r c u l i n p o s i t i v e subject or m e a n + 1 . 5 2 S.D. f r o m a ne-
1.2
x Z Z O
_o
•
Mononuclear
O
Buffy coat
cells cells
m
RN PPD+ve
[~
PPD-ve
AD
JS
l;W
Fig. 5. Comparison of the Migration Indices of 15 subjects simultaneously obtained with preparations of mononuclear cells and buffy coat cells. Fig. 6. Migration Indices of 4 subjects repeated on different days using mononuclear cells.
gative subject is still < 0 . 7 3 or ~ 0 . 7 3 respectively, this test will give 6 false negatives out of 100 positive subjects, and also 6 false positives out of 100 negative subjects. That there would be overlaps in the results is unavoidable since the tuberculin test is not an all or none phenomenon, and while someone who is negative to 10 tuberculin units may be positive to a higher concentration of tuberculin, another may be positive to 10 tuberculin units but negative to a smaller concentration of tuberculin. Using mixed leukocyte preparations (fig. 2) the MI for the group of tuberculin positive subjects was 0.69 + 0.26 (n = 19) and for the negative subjects was 0.76 +- 0.24 (n = 10). The MI were not significantly different from each other even though the mean for the positive group was less than t h a t for the negative group. Using mixed leukocyte preparations, the degree of inhibition showed no correlation with the concentration of the antigen used (fig. 3), whereas with mononuclear cell preparations, the MI showed an inverse correlation with the concentration of tuberculin used (fig. 4). Fig. 5 illustrates the MI obtained simultaneously using mononuclear cells, and buffy coat cells from 11 tuberculin positive subjects and 4 tuberculin imgative subjects. It can be seen t h a t there are 4 false negative and 2 false positive results with buffy coat cells but none with mononuclear cells. Using mixed leukocyte preparations, wide daily variations have been noted (Maini et al., 1973) and the results were not reproducible. However, the mononuclear cells migration inhibition test was shown to be more reproducible from day to day (fig. 6). Subject R.N. has an MI of 0.55 + 0.06 (mean +S.E.) and subject F.W., 0.96 + 0.03. Bull et al. (1973) also found that using preparations of mononuclear cells, the migration inhibition test was more reproducible than using preparations of mixed leukocytes with a high percentage of polymorphs. Furthermore, the coefficient of variation for the mixed leukocyte migration inhibition test was nearly double that for the mononuclear migration inhibition test and so even between replicates, the former test is less reproducible t h a t the latter. Our results using b u f f y coat cells were similar to those of Kaltreider et al. (1969). However, our results with mononuclear cells seemed to contradict those of Zabriskie and Falk (1970). We suggest that this discrepancy may be due to technical differences. They used a sedimentation m e t h o d with gelatin for collecting mononuclear cells, and also, the m a x i m u m concentration of PPD antigen used was 50 pg/ml. It is of interest to note that on three occasions when we used 50 pg/ml of PPD the MI were more than 0.73 (0.77, 0.81 and 0.98) even though the subjects demonstrated significant inhibitions at 100 ug/ml PPD (fig. 4). Objections may be raised to our using such a high concentration of antigen, but using the trypan blue exclusion test, we have shown that 90--95% of the cells were still viable after an 18 h incubation. There are two possible reasons for the failure to establish a satisfactory correlation between mixed leukocyte migration inhibition and the tuberculin reaction. The m e t h o d of collecting leukocytes by allowing red cells to settle
s p o n t a n e o u s l y f o r one h o u r and t a k i n g t h e l e u k o c y t e rich p l a s m a can n e v e r b e r e p r o d u c i b l e since d i f f e r e n t subjects h a v e d i f f e r e n t s e d i m e n t a t i o n rates, and f u r t h e r , q u i t e a large p r o p o r t i o n o f w h i t e cells m u s t i n v a r i a b l y be l e f t behind. S e c o n d l y , with b u f f y c o a t p r e p a r a t i o n s , i n v a r i a b l y t h e r e w o u l d be a b o u t 3 0 - - 5 0 % c o n t a m i n a t i o n w i t h red cells, a n d as red cells p r o b a b l y do n o t r e s p o n d to l y m p h o k i n e s , t h e y o f t e n m a s k the area o f m i g r a t i o n o f the l e u k o c y t e s . On f o u r occasions w i t h t u b e r c u l i n positive subjects, w h e n o u r m o n o n u c l e a r p r e p a r a t i o n s were c o n t a m i n a t e d w i t h m o r e t h a n 20% p o t y m o r p h s , t h e MIs were 0.78, 0.74, 0.72 and 0.72, w h i c h o n r e p e a t w h e n the p r e p a r a t i o n s h a v e less t h a n 5% p o l y m o r p h s , the MIs w e r e 0.54, 0.54, 0.38 a n d 0.51 respectively. We c o n c l u d e t h e r e f o r e t h a t t h e m i g r a t i o n i n h i b i t i o n t e s t using m o n o n u clear cells is a quick, simple and i n e x p e n s i v e i m m u n o l o g i c a l t e s t w h i c h has an excellent correlation with delayed hypersensitivity.
REFERENCES Bloom, B.R. and B. Bennett, 1968, Fed. Proc. 27, 13. Boyum, A., 1968, Scand. J. Clin. Lab. Invest. 21 suppl., 97. Bull, D.M., J.R. Leibach, M.A. Williams and R.A. Helms, 1973, Science 181,957. David, J.R., S. A1-Askari, H.S. Lawrence and L.J. Thomas, 1964, J. Immunol. 93, 264. Federlin, K., R.N. Maini, D.S. Russell and D.C. Dumonde, 1971, J. Clin. Pathol. 24,533. Gorski, A.J., 1974, Clin. Exp. Immunol. 18,149. Kaltreider, H.B., D. Soghor, J.B. Taylor and J.L. Decker, 1969, J. Immunol. 103, 179. Lockshin, M.D., 1969, Proc. Soc. Exp. Biol. Med. 132, 928. Maini, R.N., L.M. Roffe, I.T. Magrath and D.C. Dumonde, 1973, Int. Arch. Allergy 45, 308. Mitchell, C.G., M.G.M. Smith, P.L. Golding, A.L.W.F. Eddleston and R. Williams, 1972, Clin. Exp. Immunol. 11,535. Rocklin, R.E., O.L. Meyers and J.R. David, 1970, J. Immunol. 104, 95. Rosenberg, S.A. and J.R. David, 1970, J. Immunol. 105, 1447. S~bborg, M. and G. Bendixen, 1967, Acta Med. Scand. 181,247. S~bborg, M., 1967, Acta Med. Scand. 182, 167. Thor, D.E., K.E. Jureziz, S.R. Veach, E. Miller and S. Dray, 1968, Nature 219, 755. Zabriskie, J.B. and R.E. Falk, 1970, Nature (Lond.) 226,943.
1
© N o r t h - H o l l a n d P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
M O N O N U C L E A R CELLS MIGRATION INHIBITION AND D E L A Y E D HYPERSENSITIVITY IN MAN
R O N A L D P. NG a n d C O N S T A N T I ' N G. A L E X O P O U L O S
Department of Clinical Haematology, University College Hospital Medical School, London W.C.I., England (Received 9 S e p t e m b e r 1 9 7 5 , a c c e p t e d 9 N o v e m b e r 1 9 7 5 )
T h e m i g r a t i o n i n h i b i t i o n b y soluble PPD o f p e r i p h e r a l b l o o d m o n o n u c l e a r cells s h o w e d a m u c h b e t t e r r e f l e c t i o n of delayed h y p e r s e n s i t i v i t y t o t u b e r c u l i n in m a n t h a n t h e m i g r a t i o n i n h i b i t i o n of b u f f y c o a t cells. T h e m i g r a t i o n i n d e x (MI) w i t h m o n o n u c l e a r cells in a g r o u p of t u b e r c u l i n positive s u b j e c t s was 0.51 -+ 0.14 ( m e a n ± S.D.) (n = 20) and t h a t of a g r o u p of t u b e r c u l i n negative s u b j e c t s was 0.89 ± 0.11 (n = 14) (P < 0.001). Using b u f f y c o a t cells t h e MI was 0.65 -+ 0.26 (n = 19) for a g r o u p o f t u b e r c u l i n positive s u b j e c t s a n d 0 . 7 6 ± 0.24 (n = 10) for t h e g r o u p of t u b e r c u l i n negative subjects. T h e y were n o t significantly d i f f e r e n t (P > 0.05). T h e m o n o n u c l e a r cells m i g r a t i o n i n h i b i t i o n was also q u i t e r e p r o d u c i b l e f r o m day to day.
The Macrophage Migration Inhibition test is an accepted in vitro reflection of delayed hypersensitivity in guinea pigs (David et al., 1964; Bloom and Bennet, 1968). A similar technique using human l y m p h o c y t e culture supernatant and guinea pig peritoneal macrophages has also been developed {Thor et al., 1968; Rocklin et al., 1970). Although this indirect m e t h o d showed a good correlation with in vivo tests for delayed hypersensitivity, it could not be widely used in clinical situations because of its technical complexity. However, an in vitro m e t h o d (SOborg and Bendixen, 1967) based on inhibition of the migration of human peripheral blood b u f f y coat cells in the presence of Brucella antigen has been found to correlate well with skin test results (SCborg, 1967). A review of the literature since then shows that there are a number of m e t h o d s for doing such an in vitro test, using soluble or particulate antigens. When soluble antigens were used some investigators failed to show any correlation between the results of the test and delayed hypersensitivity in man (Kaltreider et al., 1969; Lockshin, 1969), while others could do so only with considerable effort and care (Rosenberg et al., 1970; Federlin et al., 1971; Mitchell et al., 1972). Several workers have suggested that only particulate antigens could given reliable results (Federlin et al., 1971; Gorski, 1974). Most authors measured the entire area of migration covered b y the cells; however Rosenberg and David (1974) found that only the inner dense area of migration showed inhibition by specific antigen in sensitive individuals, while the 'halo' of surrounding cells did n o t show in-
hibition of migration. They pointed out that about 90% of the cells in the 'halo' were polymorphs and only a few mononuclears were present. SOborg and Bendixen (1967) also found that the cells involved in the leukocytes Migration Inhibition Test were mononuclear cells. Because of our interest in clinical applications of the test, and in view of the discrepancies reported in the literature, we have investigated the relationship between the Leukocyte Migration Inhibition Test and delayed hypersensitivity in man using a soluble purified protein derivative (PPD) as the antigen together with pure preparations of mononuclear cells, and compared the results obtained with those using buffy coat preparations containing both mononuclear cells and polymorphs. MATERIALS AND METHODS
Subjects Thirty-two healthy adult volunteers, whose reaction to 10 units of tuberculin (T.U.) had recently been tested, were selected for study. In ten instances, blood from the umbilical cord of new born infants {assumed to be tuberculin negative), was used.
Preparation of cells Bully coat cells. Ten ml of blood was taken into heparinised plastic tubes and allowed to stand for 1 h at 37°C. The leukocyte rich plasma was then taken off, and centrifuged and the cells obtained were washed three times in phosphate buffered saline at pH 7.3. Mononuclear cells. Ten ml of blood was mixed with an equal volume of phosphate buffered saline and mononuclear cells (95% purity) were then obtained by centrifugation in a gradient of Ficoll--Triosil (Boyum, 1968). The cells were then washed three times in phosphate buffered saline. Migration test The preparations of buffy coat cells or mononuclear cells were taken into mmrohaematocrit tubes (internal diameter of 1 mm, and 75 m m in length) wi~ich were sealed with 'Cristaseal' and centrifuged (12,000 g) for 5 min. The tubes were cut at the interface between the fluid and the cell layer and secured into wells on plastic micro-migration plates (Sterilin). The wells were filled with a solution containing RPMI 1640 medium (Biocult) 9 vol, fetal calf serum 1 vol, Gentamycin 4 gg/ml and preservative-free PPD (Weybridge Central Veterinary Laboratory, Ministry of Agriculture) 100 pg/ml; in the control well PPD was omitted. The wells were sealed with glass cover-slips and silicone grease and placed horizontally into an incubator for 18 h at 37°C. The area of migration was projected and traced onto paper and the rel-
ative size of the projected areas measured by cutting out and weighing. All tests were carried out in triplicate. The coefficient of variation between replicates for the mononuclear cells migration inhibition test was 9.9% and that for the mixed leukocyte migration inhibition test was 18.2%. The migration areas in the presence and absence of antigen were compared and expressed as an index using the formula: Area of migration in presence of antigen Migration Index (MI) = Area of migration in absence of antigen In 15 subjects simultaneous migration tests were set up with both mononuclear cells and buffy coat cells. RESULTS AND DISCUSSION
Using mononuclear cells, the MI for a group of tuberculin positive subjects was 0.51 + 0.14 (mean + S.D.) (n = 20) and that for the tuberculin negative subjects was 0.89 + 0.11 (n = 14) and they are significantly different ( P < 0.001). The MI value of 0.73 was then chosen as the dividing point for separating the 2 groups, as 0.73 is 1.52 S.D. away from the mean of both groups.
b
A&
•
&&
h
•
&A AA
X w
.E
A & A
_'2
Z
Z
0
:e .e e:e
Z .( Z 0
0
00 oo
.4--
o •
•
0
.2D
0
•
p pD +re
•
PPD -ve
• •
PPD PPD
+re -ve
Fig. 1. Migration Indices of tuberculin positive and tuberculin negative subjects using preparations of mononuclear cells. Fig. 2. Migration Indices of tuberculin positive and tuberculin negative subjects using preparations of buffy coat cells.
1.0
I.O
:E × w c~ Z
× w z
z 0
Z 0.5
..5
1 50
I 1OO
I 150
I
I 200
50
PPD pg/ml
I
IOO
I
I
150
200
PPD pg,'nq
Fig. 3. Migration Indices of 5 tuberculin positive subjects using preparations of buffy coat cells at different concentrations of PPD. Fig. 4. Migration Indices of 5 tuberculin positive subjects using mononuclear cells at different concentrations of PPD.
As can be seen f r o m fig. 1, there w a s n o overlap b e t w e e n t h e groups o f subjects studied. On a t h e o r e t i c a l basis a standard score (or z score) o f + 1 . 5 2 inc l u d e s 87% o f t h e p o p u l a t i o n and since a n y value that is less than m e a n - - 1 . 5 2 S.D. f r o m a t u b e r c u l i n p o s i t i v e subject or m e a n + 1 . 5 2 S.D. f r o m a ne-
1.2
x Z Z O
_o
•
Mononuclear
O
Buffy coat
cells cells
m
RN PPD+ve
[~
PPD-ve
AD
JS
l;W
Fig. 5. Comparison of the Migration Indices of 15 subjects simultaneously obtained with preparations of mononuclear cells and buffy coat cells. Fig. 6. Migration Indices of 4 subjects repeated on different days using mononuclear cells.
gative subject is still < 0 . 7 3 or ~ 0 . 7 3 respectively, this test will give 6 false negatives out of 100 positive subjects, and also 6 false positives out of 100 negative subjects. That there would be overlaps in the results is unavoidable since the tuberculin test is not an all or none phenomenon, and while someone who is negative to 10 tuberculin units may be positive to a higher concentration of tuberculin, another may be positive to 10 tuberculin units but negative to a smaller concentration of tuberculin. Using mixed leukocyte preparations (fig. 2) the MI for the group of tuberculin positive subjects was 0.69 + 0.26 (n = 19) and for the negative subjects was 0.76 +- 0.24 (n = 10). The MI were not significantly different from each other even though the mean for the positive group was less than t h a t for the negative group. Using mixed leukocyte preparations, the degree of inhibition showed no correlation with the concentration of the antigen used (fig. 3), whereas with mononuclear cell preparations, the MI showed an inverse correlation with the concentration of tuberculin used (fig. 4). Fig. 5 illustrates the MI obtained simultaneously using mononuclear cells, and buffy coat cells from 11 tuberculin positive subjects and 4 tuberculin imgative subjects. It can be seen t h a t there are 4 false negative and 2 false positive results with buffy coat cells but none with mononuclear cells. Using mixed leukocyte preparations, wide daily variations have been noted (Maini et al., 1973) and the results were not reproducible. However, the mononuclear cells migration inhibition test was shown to be more reproducible from day to day (fig. 6). Subject R.N. has an MI of 0.55 + 0.06 (mean +S.E.) and subject F.W., 0.96 + 0.03. Bull et al. (1973) also found that using preparations of mononuclear cells, the migration inhibition test was more reproducible than using preparations of mixed leukocytes with a high percentage of polymorphs. Furthermore, the coefficient of variation for the mixed leukocyte migration inhibition test was nearly double that for the mononuclear migration inhibition test and so even between replicates, the former test is less reproducible t h a t the latter. Our results using b u f f y coat cells were similar to those of Kaltreider et al. (1969). However, our results with mononuclear cells seemed to contradict those of Zabriskie and Falk (1970). We suggest that this discrepancy may be due to technical differences. They used a sedimentation m e t h o d with gelatin for collecting mononuclear cells, and also, the m a x i m u m concentration of PPD antigen used was 50 pg/ml. It is of interest to note that on three occasions when we used 50 pg/ml of PPD the MI were more than 0.73 (0.77, 0.81 and 0.98) even though the subjects demonstrated significant inhibitions at 100 ug/ml PPD (fig. 4). Objections may be raised to our using such a high concentration of antigen, but using the trypan blue exclusion test, we have shown that 90--95% of the cells were still viable after an 18 h incubation. There are two possible reasons for the failure to establish a satisfactory correlation between mixed leukocyte migration inhibition and the tuberculin reaction. The m e t h o d of collecting leukocytes by allowing red cells to settle
s p o n t a n e o u s l y f o r one h o u r and t a k i n g t h e l e u k o c y t e rich p l a s m a can n e v e r b e r e p r o d u c i b l e since d i f f e r e n t subjects h a v e d i f f e r e n t s e d i m e n t a t i o n rates, and f u r t h e r , q u i t e a large p r o p o r t i o n o f w h i t e cells m u s t i n v a r i a b l y be l e f t behind. S e c o n d l y , with b u f f y c o a t p r e p a r a t i o n s , i n v a r i a b l y t h e r e w o u l d be a b o u t 3 0 - - 5 0 % c o n t a m i n a t i o n w i t h red cells, a n d as red cells p r o b a b l y do n o t r e s p o n d to l y m p h o k i n e s , t h e y o f t e n m a s k the area o f m i g r a t i o n o f the l e u k o c y t e s . On f o u r occasions w i t h t u b e r c u l i n positive subjects, w h e n o u r m o n o n u c l e a r p r e p a r a t i o n s were c o n t a m i n a t e d w i t h m o r e t h a n 20% p o t y m o r p h s , t h e MIs were 0.78, 0.74, 0.72 and 0.72, w h i c h o n r e p e a t w h e n the p r e p a r a t i o n s h a v e less t h a n 5% p o l y m o r p h s , the MIs w e r e 0.54, 0.54, 0.38 a n d 0.51 respectively. We c o n c l u d e t h e r e f o r e t h a t t h e m i g r a t i o n i n h i b i t i o n t e s t using m o n o n u clear cells is a quick, simple and i n e x p e n s i v e i m m u n o l o g i c a l t e s t w h i c h has an excellent correlation with delayed hypersensitivity.
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