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PPD-induced lymphocyte transformation in vitro using whole blood
CLINICAL
IMML’NOLOGY
IN”
IMMUNOPATHOLOGY
PPD-Induced
8,
Lymphocyte Transformation Using Whole Blood’ M. K. VILJANEX
Department
28- 33 (1977)
of Medicul
Microbiologv.
Turhr
AND
J.
in Vitro
ESKOLA
Uniwrsity.
St;-20520
TurXu
52. Firdtrrrd
Received July 9. 1976 A simple micromethod for stimulation of human lymphocytes with purified protein derivative of tuberculin (PPD) was developed. The use of small amounts of whole blood, microtiter plates, [5-rz51]iodo-2’-deoxyuridine together with Sfluoro-2’-deoxyuridine and a multiple cell-culture harvester makes the method easy, rapid, and suitable for routine laboratory use. As an application, the results obtained in this test were compared to the tuberculin skin test. A significantly higher response of lymphocytes to PPD was found in persons positive to 1 TU in the skin test than in persons positive to 10 TU. No correlation between the diameter of induration in the skin test and lymphocyte transformation was observed within these groups.
INTRODUCTION
The tuberculin skin test has been used routinely as a parameter of immunity against tuberculosis. Since there is some controversy concerning the reliability of the tuberculin skin test, in vitro methods have been developed to assess cellmediated immune reactions against purified protein derivative of tuberculin (PPD) (l-4). The controversial findings regarding the correlation between the skin test and the leukocyte-migration inhibition test with PPD have cast some doubt on the usefulness of this test as an in vitro parameter of delayed hypersensitivity (5-8). In contrast, several authors have obtained a good correlation between the skin test and the lymphocyte transformation test with PPD (8- 13). The conventional methods for lymphocyte stimulation are cumbersome, timeconsuming, and require large blood samples. Therefore, many microtechniques for PPD stimulation have been reported. Among these, the techniques using whole blood (14-18) probably correlate better to in \v!vo conditions than those using isolated lymphocytes. At the same time they measure the effect of plasma factors and cells other than lymphocytes affecting the response (19,20). Recently, we have developed a simple micromethod to measure the response of lymphocytes against mitogens (21). This technique utilizes whole blood, microtiter plates, [.5‘251]iodo-2’-deoxyuridine ( 1251UdR) together with 5-fluoro-2’-deoxyuridine (FUdR) in place of r3H-lthymidine, and a multiple cell-culture harvester. In this work, the same principle has been applied to lymphocyte stimulation with PPD and, as an application, the results obtained in this test have been correlated to tuberculin skin tests. MATERIALS
AND METHODS
Subjects Healthy male medical students and members of the laboratory 1 This study was supported by a grant from the Finnish Anti-Tuberculosis 28 Copyright All
rights
0
1977
of reproduction
by
Academic in any
Press,
Inc.
form
resewed.
staff, aged 20-30
Association.
I.S.SN
011911L113
WHOLE-BLOOD
CULTURE
FOR
PPD
STIMULATION
29
years, served as blood donors. All of them had been vaccinated neonatally and boostered at the age of lo-15 years with Bacillus-Calmette-Guerin (BCG). They were skin tested first with 1 TU of PPD and in negative cases (diameter of induration <6 mm) skin testing was repeated with 10 TU of PPD. The persons unresponsive to 10 TU were revaccinated with BCG 2- 16 weeks before the lymphocyte transformation test. The skin tests were performed at least 20 weeks before the in vitro studies. Blood Blood (0.5 ml) was obtained by venipuncture and put immediately into sterile culture tubes (2001, Falcon Plastics, Los Angeles, Calif.) containing 25 units of preservative-free heparin. PPD PPD tuberculin in RT 23 (Statens Seruminstitut, Copenhagen, Denmark) was used throughout the study. To attain the desired concentrations of PPD, the stock solution (2 mg/ml) was diluted immediately prior the experiments in RPM1 1640 (Grand Island Biological Co., Grand Island, N.Y.) containing 100 units/ml of penicillin and 100 pg/rnl of streptomycin. Culture Conditions Our routine method for lymphocyte stimulation with mitogens (21) was used with slight modifications. In brief, a mixture of 25 ~1 of heparinized blood and 75 ~1 of RPM1 1640 containing antibiotics was placed into each well of a microtiter plate (IS-MRC-96, Linbro Chemical Co., New Haven, Conn.). Each well contained, thus, 25-150 x lo3 lymphocytes and 2.5-30 x IO3 monocytes. These figures were calculated from the normal values for differential leukocyte counts in the Finnish population. Thereafter, PPD in 25 ,ul of RPM1 1640 was added into the desired wells. Control cultures received 25 ~1 of plain medium. All cultures were prepared in quadruplicate. The plates were incubated for various times in humidified air and 5% CO, at 37°C. Assessment of the Response To measure the degree of lymphocyte transformation, 20 ~1 of t251UdR (sp act, l-6 mCi/mg; The Radiochemical Centre, Amersham, England) was added into each well together with FUdR (Fluka, Buchs, Switzerland) to a final concentration of lop6 M (22). The cultures were harvested with a multiple cell-culture harvester (Skatron, Lierbyen, Norway) and the radioactivities were measured by an automatic well-type y-counter (LKB-Wallac, Turku, Finland). The responses are expressed as counts per minute or as stimulation indices, i.e., the ratio between cpm of the stimulated cultures and cpm of the control cultures. RESULTS
Dose of lz511JdR and Incorporation Time In preliminary experiments, two doses of lzsIUdR, 0.125 and 0.250 $X/culture, were tested. Since the larger amount resulted in better reproducibility in the control cultures, it was chosen for further experiments. The incorporation of 1251UdR reached maximal and balanced level at 14 hr. For practical reasons, an incorporation time of 18 hr was used in further studies.
30
VILJANES
ANI~
ESliOL.4
Concentration of PPD and Stimulation Time Five concentrations of PPD were used: 0.01, 0.1, 1.0, 10, and 100 pg/ml. Peak response was achieved with all the concentrations used at 90 hr of total incubation (Fig. 1). PPD concentrations of 10 and 100 pg/ml elicited maximal and equal responses in persons with both high and low responses. The former responded well to the lowest concentrations of PPD, while the latter responded only to the concentrations over 1.O pg/ml. In further studies, PPD concentrations of 1.O and 100 &ml were used. Optimal Conditions jiw the Method The following conditions were found most suitable for testing the response of lymphocytes to PPD: 25 ~1 of heparinized blood plus 100 ~1 of RPM1 1640 containing 0.125 and 12.5 pg of PPD (corresponding final concentrations of 1.O and 100 p.g/ml, respectively); incubation on microtiter plates for 72 hr at 37°C with 5% CO,; and the addition of 0.25 &i of lz51UdR with FUdR (final concentration of lo-” M) per culture for a final 18 hr of incubation. Correlation betllvell the Lymphocyte Tmnsj~~rrntrtioll ulld Skin Tests In total, 85 persons were tested. Fifty-eight of them had positive skin tests to 1 TU, 20 persons had positive skin reactions to 10 TU. and 7 persons were skin-test negative and were revaccinated with BCG 2- 16 weeks before the lymphocyte transformation test. 150
a
100
-
100 :.Ll~l
;5:,&&&
0
go:.]
I
42
66 INCUBATION
FIG. I, Effect incubation times
90
1U TIME
(HOURS)
of different PPD concentrations (0.01, 0.1, 1.0, 10, and 100 &ml) and different on the uptake of 12JIUdR in persons with high (a) and low (b) responses.
WHOLE-BLOOD
CULTURE
FOR
PPD
31
STIMULATION
The mean stimulation indices, both with 1.O and 100 pg/ml of PPD, of the group positive to 1 TU were significantly higher than the corresponding figures of the group positive to 10 TU (Table 1, Fig. 2). Also the mean counts of radioactivity obtained from the stimulated cultures in these groups differed significantly from each other. No clear correlation between the diameter of induration in the skin tests and lymphocyte transformations was observed within these groups (Table 2). TABLE
1
LYMPHOCYTE TRANSFORMATION IN VITRO TO STIMULATION WITH PPD IN DIFFERENT SKIN-REACTIVE GROUP*
Group
PPD (1 &ml)
PPD (100 &ml)
Number of donors
wm
Stimulation index
wm
Stimulation index
58 20
1243 & 1019 600 k 366*
22.0 ? 23.2 7.8 k 4.6*
1020 2 634 573 ” 378*
16.6 Ifr 12.0 6.9 k 4.1**
7
506 2 332
548 k 272
6.4 + 2.8
positive positive 10-TU negative, revaccinated I-TU IO-TU
6.0 k
3.6
n Mean -c SD are given. * p < 0.01 when compared to respective values of the I-TU positive group (Student’s t test). ** p < 0.001 when compared to respective values of the l-TU positive group (Student’s r test). 12
. 11 %
. . 9I
.
60-
. 50-
. .
:: P .t 40.
..
s ; E z 30Z YI 201
10.
.. a
. . I
.
i . w . I.. . .& 0 v.-.* . ::.. .. .
. : -#n-.
. . .. ,A. .:*. : *
. -7’
4 :::
i
0' ,.,‘s,:,~e
,,~~,~$evaccinated
PPD 1 vg/ml
’ T” lo T” revaccinated positive porltlve
PPD 100 rg/ml
2. Individual stimulation indices obtained by two concentrations of PPD (1 and 100 CLg/ml) in different skin-reactive groups. Mean values are indicated by solid lines and SD values by dotted lines. FIG.
PPD (&ml)
Group l-TU IO-TU
1 100 I 100
positive positive
” Correlation coefficient. li Probability by Student’s I’ Not significant, N.S.
.. 0.06 -0.02 0.45 0.42
N.S. N.S. N.S. N.S.
I test.
In the BCG-revaccinated group, the lymphocyte transformation induced by PPD was of the same magnitude as in the group positive to 10 TU (Table 1, Fig. 2). DISCUSSION
Several factors decrease the reliability of the tuberculin skin test, e.g., nonspecific skin reactions, inadequate injection techniques, and inaccurate quantitation of the indurations. Therefore, many in rirro methods have been developed to assess cell-mediated immunity against tuberculosis. Since lymphocyte transformation induced by PPD has been found in many studies to correlate well to the skin test (8-13), a simple and reliable method is needed for the lymphocyte transformation test. In this report, we describe a micromethod for lymphocyte stimulation with PPD suitable for routine use. Since whole blood is used in this method, the thymus-derived lymphocytes and other cells and serum factors affecting the response (19,20) are taken into account. The utilization of small volumes of blood in microplates and of a multiple cell-culture harvester enables the simultaneous assay of a high number of samples. The use of lz51UdR makes the counting of radioactivities easy and rapid (22,23). With a suitable amount of FUdR to enhance the incorporation of ““IUdR, stimulation indices are of the same magnitude as with [3H-]thymidine (22). Since delayed skin reactions and lymphocyte stimulation in vitro both depend upon the presence of stimulated lymphocytes, a high degree of correlation can be expected between these two tests. There are, however, conflicting results on this point. Like some other authors (8- 13) we have in the present study been able to demonstrate a significant correlation between the lymphocyte transformation and skin tests, although there is wide overlapping in the individual values of different groups. The negative correlation between these tests reported by some authors (1,24-28) may be due to the differences in the experimental techniques. The purification of lymphocytes may exclude the adjacent cells possibly participating in the in \,ifro lymphocyte transformation process. The enhancing effect of monocytes on the stimulation of lymphocytes with antigens (29-31) and mitogens (32-24) has been reported earlier. Thus, the method using whole blood can be considered to have a good correlation to in vivo events. High concentrations of PPD have been reported to induce transformation of lymphocytes from tuberculin-negative donors, possibly due to a polyclonal B-cell
WHOLE-BLOOD
CULTURE
FOR
PPD
33
STIMULATION
activation (11). However, the B-cell mitogenicity of PPD has been demonstrated by spleen cells from mice and guinea pigs (35,36), but is not confirmed in man. The in vitro response to high doses of this antigen could be expected to be less clearly related to the skin test than the in vitro response to lower doses. Our results do not confirm this concept, since with both low (0.125 pg) and high (12.5 pg) doses of PPD, equal and good relationships can be obtained between the lymphocyte transformation and skin testing. This disagreement may be due to the requirement of higher doses of antigen in the method using whole blood. Concentrations of PPD exceeding 100 PgIrnl may be needed to demonstrate the possible nonspecific mitogenicity of PPD in the present system. REFERENCES 1. Pearmain, G., Lycette, R. R., and Fitzgerald, P. H., Lancer 1, 637, 1963. 2. Schreck, R., Amer. Rev. Resp. Dis. 87, 734, 1963. 3. Clausen, J. E., and Soborg, M., Acta Med. Stand. 186, 227, 1969. 4. Clausen, J. E., Acta Allerg. (Kbh.) 26, 56, 1971. 5. Lockshin, M. D., Proc. Sot. Exp. Biol. Med. 132, 928, 1969. 6. Kaltreider, H. B., Soghor, D., Taylor, J. B., and Decker, J. L., J. Immunol. 103, 179, 1969. 7. Rosenberg, S., and David, J. R., J. Immunol. 105, 1447, 1970. 8. Fleer, A., van der Hart, M., Blok-Schut, B. J. Th., and Schellekens, P. Th. A., Eur. J. Immunol. 6, 163, 1976. 9. Kerby, G. R., Amer. Rev. Resp. Dis. 97, 904, 1968. 10. Hinz, C. F., Jr., Daniel, T. M., and Baum, G. L.. Znt. Arch. Allergy 38, 119, 1970. Il. Nilsson, B. S., Cell. Immunol. 3, 493, 1972. 12. Miller, S. D., and Jones, H. E., Amer. Rev. Resp. Dis. 107, 503, 1973. 13. Gloss, O., Infect. Immun. 11, 1163, 1975. 14. Junge, U., Hoekstra, J., Wolfe, L., and Deinhardt, F., Clin. Exp. fmmunol. 7, 431, 1970. 15. Paty, D. W., and Hughes, D., J. Immunol. Method. 2, 99, 1972. 16. Han, T., and Pauly, J., Clin. Exp. Immunol. 11, 137, 1972. 17. Pauly, J. L., Sokal, J. E., and Han, T., J. Lab. Clin. Med. 82, 500, 1973. 18. Condoulis, W. V., Soltysik, L. M., and Baram, P., J. Med. Prim. 3, 251, 1974. 19. Park. B. H., and Good, R. A., Proc. Nat. Acad. Sci. U.S. 69, 371, 1972. 20. Lopez, C., Simmons, R. L., Park, B. H., Najarian, J. S., and Good, R. A., Clin. Exp. Zmmunol. 16, 565, 1974.
21. Eskola, J., Soppi, E., Viljanen, M., and Ruuskanen, O., Immunol. Commun. 4, 297, 1975. 22. Asantila, T., and Toivanen, P., J. Immunol. Method. 6, 73, 1974. 23. Pellegrino, M. A., Ferrone, S., Pellegrino, A., and Reisfeld, R. A., Clin. Immunol. munopathol.
Im-
2, 67, 1973.
24. Aspergren, N., and Rorsman, H., J. Allergy 35, 433, 1964. 25. McFarland, W., and Heilman, D., Amer. Rev. Resp. Dis. 93, 742, 1%6. 26. Gump, D. W., Fekety, R., Jr., Urbanetti, J., and Nosenzo, C., Amer. Rev. Resp. Dis. 95, 470, 1967. 27. Matsaniotis, N., Tsenghi, C., Economou-Mavrou, C., and Mataxotou-Stavridaki, C., J. Pediar. 72, 599, 1968. 28. Thomas, J. W., Clements, D., and Grzybowski, S., Clin. Exp. Immunol. 9, 611, 1971. 29. Oppenheim, J. J., Leventhal, B. G., and Hersh, E. M., J. Zmmunol. 101, 262, 1968. 30. Hersh, E. M., and Harris, J. E., J. Immunol. 100, 1184, 1968. 31. Seeger, R. C., and Oppenheim, J. J., J. Immunol. 109, 244, 1972. 32. Hedfors, E., Helm, G., and Pettersson, D., Ctin. Exp. lmmunol. 22, 223, 1975. 33. Rosenstreich, D. L., Farrar, J. J., and Dougherty, S., J. Immunol. 116, 131, 1976. 34. Schmidke, J. R., and Hatfield, S., J. Immunol. 116, 357, 1976. 35. Sultzer, B. M., and Nilsson, B. S., Nature Nens Biol. 240, 198, 1972. 36. Gronowicz, E., and Coutinho, A., Eur. J. Immunol. 4, 771, 1974.
IMML’NOLOGY
IN”
IMMUNOPATHOLOGY
PPD-Induced
8,
Lymphocyte Transformation Using Whole Blood’ M. K. VILJANEX
Department
28- 33 (1977)
of Medicul
Microbiologv.
Turhr
AND
J.
in Vitro
ESKOLA
Uniwrsity.
St;-20520
TurXu
52. Firdtrrrd
Received July 9. 1976 A simple micromethod for stimulation of human lymphocytes with purified protein derivative of tuberculin (PPD) was developed. The use of small amounts of whole blood, microtiter plates, [5-rz51]iodo-2’-deoxyuridine together with Sfluoro-2’-deoxyuridine and a multiple cell-culture harvester makes the method easy, rapid, and suitable for routine laboratory use. As an application, the results obtained in this test were compared to the tuberculin skin test. A significantly higher response of lymphocytes to PPD was found in persons positive to 1 TU in the skin test than in persons positive to 10 TU. No correlation between the diameter of induration in the skin test and lymphocyte transformation was observed within these groups.
INTRODUCTION
The tuberculin skin test has been used routinely as a parameter of immunity against tuberculosis. Since there is some controversy concerning the reliability of the tuberculin skin test, in vitro methods have been developed to assess cellmediated immune reactions against purified protein derivative of tuberculin (PPD) (l-4). The controversial findings regarding the correlation between the skin test and the leukocyte-migration inhibition test with PPD have cast some doubt on the usefulness of this test as an in vitro parameter of delayed hypersensitivity (5-8). In contrast, several authors have obtained a good correlation between the skin test and the lymphocyte transformation test with PPD (8- 13). The conventional methods for lymphocyte stimulation are cumbersome, timeconsuming, and require large blood samples. Therefore, many microtechniques for PPD stimulation have been reported. Among these, the techniques using whole blood (14-18) probably correlate better to in \v!vo conditions than those using isolated lymphocytes. At the same time they measure the effect of plasma factors and cells other than lymphocytes affecting the response (19,20). Recently, we have developed a simple micromethod to measure the response of lymphocytes against mitogens (21). This technique utilizes whole blood, microtiter plates, [.5‘251]iodo-2’-deoxyuridine ( 1251UdR) together with 5-fluoro-2’-deoxyuridine (FUdR) in place of r3H-lthymidine, and a multiple cell-culture harvester. In this work, the same principle has been applied to lymphocyte stimulation with PPD and, as an application, the results obtained in this test have been correlated to tuberculin skin tests. MATERIALS
AND METHODS
Subjects Healthy male medical students and members of the laboratory 1 This study was supported by a grant from the Finnish Anti-Tuberculosis 28 Copyright All
rights
0
1977
of reproduction
by
Academic in any
Press,
Inc.
form
resewed.
staff, aged 20-30
Association.
I.S.SN
011911L113
WHOLE-BLOOD
CULTURE
FOR
PPD
STIMULATION
29
years, served as blood donors. All of them had been vaccinated neonatally and boostered at the age of lo-15 years with Bacillus-Calmette-Guerin (BCG). They were skin tested first with 1 TU of PPD and in negative cases (diameter of induration <6 mm) skin testing was repeated with 10 TU of PPD. The persons unresponsive to 10 TU were revaccinated with BCG 2- 16 weeks before the lymphocyte transformation test. The skin tests were performed at least 20 weeks before the in vitro studies. Blood Blood (0.5 ml) was obtained by venipuncture and put immediately into sterile culture tubes (2001, Falcon Plastics, Los Angeles, Calif.) containing 25 units of preservative-free heparin. PPD PPD tuberculin in RT 23 (Statens Seruminstitut, Copenhagen, Denmark) was used throughout the study. To attain the desired concentrations of PPD, the stock solution (2 mg/ml) was diluted immediately prior the experiments in RPM1 1640 (Grand Island Biological Co., Grand Island, N.Y.) containing 100 units/ml of penicillin and 100 pg/rnl of streptomycin. Culture Conditions Our routine method for lymphocyte stimulation with mitogens (21) was used with slight modifications. In brief, a mixture of 25 ~1 of heparinized blood and 75 ~1 of RPM1 1640 containing antibiotics was placed into each well of a microtiter plate (IS-MRC-96, Linbro Chemical Co., New Haven, Conn.). Each well contained, thus, 25-150 x lo3 lymphocytes and 2.5-30 x IO3 monocytes. These figures were calculated from the normal values for differential leukocyte counts in the Finnish population. Thereafter, PPD in 25 ,ul of RPM1 1640 was added into the desired wells. Control cultures received 25 ~1 of plain medium. All cultures were prepared in quadruplicate. The plates were incubated for various times in humidified air and 5% CO, at 37°C. Assessment of the Response To measure the degree of lymphocyte transformation, 20 ~1 of t251UdR (sp act, l-6 mCi/mg; The Radiochemical Centre, Amersham, England) was added into each well together with FUdR (Fluka, Buchs, Switzerland) to a final concentration of lop6 M (22). The cultures were harvested with a multiple cell-culture harvester (Skatron, Lierbyen, Norway) and the radioactivities were measured by an automatic well-type y-counter (LKB-Wallac, Turku, Finland). The responses are expressed as counts per minute or as stimulation indices, i.e., the ratio between cpm of the stimulated cultures and cpm of the control cultures. RESULTS
Dose of lz511JdR and Incorporation Time In preliminary experiments, two doses of lzsIUdR, 0.125 and 0.250 $X/culture, were tested. Since the larger amount resulted in better reproducibility in the control cultures, it was chosen for further experiments. The incorporation of 1251UdR reached maximal and balanced level at 14 hr. For practical reasons, an incorporation time of 18 hr was used in further studies.
30
VILJANES
ANI~
ESliOL.4
Concentration of PPD and Stimulation Time Five concentrations of PPD were used: 0.01, 0.1, 1.0, 10, and 100 pg/ml. Peak response was achieved with all the concentrations used at 90 hr of total incubation (Fig. 1). PPD concentrations of 10 and 100 pg/ml elicited maximal and equal responses in persons with both high and low responses. The former responded well to the lowest concentrations of PPD, while the latter responded only to the concentrations over 1.O pg/ml. In further studies, PPD concentrations of 1.O and 100 &ml were used. Optimal Conditions jiw the Method The following conditions were found most suitable for testing the response of lymphocytes to PPD: 25 ~1 of heparinized blood plus 100 ~1 of RPM1 1640 containing 0.125 and 12.5 pg of PPD (corresponding final concentrations of 1.O and 100 p.g/ml, respectively); incubation on microtiter plates for 72 hr at 37°C with 5% CO,; and the addition of 0.25 &i of lz51UdR with FUdR (final concentration of lo-” M) per culture for a final 18 hr of incubation. Correlation betllvell the Lymphocyte Tmnsj~~rrntrtioll ulld Skin Tests In total, 85 persons were tested. Fifty-eight of them had positive skin tests to 1 TU, 20 persons had positive skin reactions to 10 TU. and 7 persons were skin-test negative and were revaccinated with BCG 2- 16 weeks before the lymphocyte transformation test. 150
a
100
-
100 :.Ll~l
;5:,&&&
0
go:.]
I
42
66 INCUBATION
FIG. I, Effect incubation times
90
1U TIME
(HOURS)
of different PPD concentrations (0.01, 0.1, 1.0, 10, and 100 &ml) and different on the uptake of 12JIUdR in persons with high (a) and low (b) responses.
WHOLE-BLOOD
CULTURE
FOR
PPD
31
STIMULATION
The mean stimulation indices, both with 1.O and 100 pg/ml of PPD, of the group positive to 1 TU were significantly higher than the corresponding figures of the group positive to 10 TU (Table 1, Fig. 2). Also the mean counts of radioactivity obtained from the stimulated cultures in these groups differed significantly from each other. No clear correlation between the diameter of induration in the skin tests and lymphocyte transformations was observed within these groups (Table 2). TABLE
1
LYMPHOCYTE TRANSFORMATION IN VITRO TO STIMULATION WITH PPD IN DIFFERENT SKIN-REACTIVE GROUP*
Group
PPD (1 &ml)
PPD (100 &ml)
Number of donors
wm
Stimulation index
wm
Stimulation index
58 20
1243 & 1019 600 k 366*
22.0 ? 23.2 7.8 k 4.6*
1020 2 634 573 ” 378*
16.6 Ifr 12.0 6.9 k 4.1**
7
506 2 332
548 k 272
6.4 + 2.8
positive positive 10-TU negative, revaccinated I-TU IO-TU
6.0 k
3.6
n Mean -c SD are given. * p < 0.01 when compared to respective values of the I-TU positive group (Student’s t test). ** p < 0.001 when compared to respective values of the l-TU positive group (Student’s r test). 12
. 11 %
. . 9I
.
60-
. 50-
. .
:: P .t 40.
..
s ; E z 30Z YI 201
10.
.. a
. . I
.
i . w . I.. . .& 0 v.-.* . ::.. .. .
. : -#n-.
. . .. ,A. .:*. : *
. -7’
4 :::
i
0' ,.,‘s,:,~e
,,~~,~$evaccinated
PPD 1 vg/ml
’ T” lo T” revaccinated positive porltlve
PPD 100 rg/ml
2. Individual stimulation indices obtained by two concentrations of PPD (1 and 100 CLg/ml) in different skin-reactive groups. Mean values are indicated by solid lines and SD values by dotted lines. FIG.
PPD (&ml)
Group l-TU IO-TU
1 100 I 100
positive positive
” Correlation coefficient. li Probability by Student’s I’ Not significant, N.S.
.. 0.06 -0.02 0.45 0.42
N.S. N.S. N.S. N.S.
I test.
In the BCG-revaccinated group, the lymphocyte transformation induced by PPD was of the same magnitude as in the group positive to 10 TU (Table 1, Fig. 2). DISCUSSION
Several factors decrease the reliability of the tuberculin skin test, e.g., nonspecific skin reactions, inadequate injection techniques, and inaccurate quantitation of the indurations. Therefore, many in rirro methods have been developed to assess cell-mediated immunity against tuberculosis. Since lymphocyte transformation induced by PPD has been found in many studies to correlate well to the skin test (8-13), a simple and reliable method is needed for the lymphocyte transformation test. In this report, we describe a micromethod for lymphocyte stimulation with PPD suitable for routine use. Since whole blood is used in this method, the thymus-derived lymphocytes and other cells and serum factors affecting the response (19,20) are taken into account. The utilization of small volumes of blood in microplates and of a multiple cell-culture harvester enables the simultaneous assay of a high number of samples. The use of lz51UdR makes the counting of radioactivities easy and rapid (22,23). With a suitable amount of FUdR to enhance the incorporation of ““IUdR, stimulation indices are of the same magnitude as with [3H-]thymidine (22). Since delayed skin reactions and lymphocyte stimulation in vitro both depend upon the presence of stimulated lymphocytes, a high degree of correlation can be expected between these two tests. There are, however, conflicting results on this point. Like some other authors (8- 13) we have in the present study been able to demonstrate a significant correlation between the lymphocyte transformation and skin tests, although there is wide overlapping in the individual values of different groups. The negative correlation between these tests reported by some authors (1,24-28) may be due to the differences in the experimental techniques. The purification of lymphocytes may exclude the adjacent cells possibly participating in the in \,ifro lymphocyte transformation process. The enhancing effect of monocytes on the stimulation of lymphocytes with antigens (29-31) and mitogens (32-24) has been reported earlier. Thus, the method using whole blood can be considered to have a good correlation to in vivo events. High concentrations of PPD have been reported to induce transformation of lymphocytes from tuberculin-negative donors, possibly due to a polyclonal B-cell
WHOLE-BLOOD
CULTURE
FOR
PPD
33
STIMULATION
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