Leukocyte Migration Test in a Modified Agarose Medium RAUNGRAIWAN KOONAKOSIT, M . S C , AND B E N C H A PETCHCLAI,
M.D.
Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Bangkok 4, Thailand
ABSTRACT
(LMT) that increases the stability of the medium has been successfully used as an in-vitro in a standard incubator. It contained 1% test of cell-mediated immunity (CMI) to agarose (Sigma Chemical Co.,), 0.003 M microbial antigens, 2 " 7 , 1 3 , 1 7 , 1 8 autoanti- HEPES (Sigma Chemical Co.), TC medium gens, 3,4,9,10,16 chemical substances, 14 and 199 (Difco), and 10% horse serum. T h e pH even tumor antigens. 1 It is also used in was adjusted to 7.3 with 2 N NaOH and mixed lymphocyte c u l t u r e reactions then 15 ml. were poured into a 9-cm. (MLC) 8 and to detect transplant rejec- plastic Petri dish. tion. 8,11 T h e agarose plate method introThe collection of heparinized blood, duced by Clausen 5 is simple and highly dextran separation of leukocytes, and reproducible. It requires smaller amounts other technics followed the method of of leukocytes and antigens and less equip- Clausen 5 and are described here briefly. ment. T h e technic should prove more useFive milliliters of blood were collected ful for clinical application if it is further into a polyethylene syringe, then transsimplified. T h e present modified medium ferred into a siliconized screw-capped can be successfully used in an ordinary tube containing 125 I.U. heparin. After incubator. dextran sedimentation and washing, leukocytes were suspended in TC 199 Materials and Methods medium (containing 10% horse serum) to make a concentration of approximately T h e method followed that of Clausen, 5 2 x 105 cells per cu. mm. For the test, and only the medium was slightly modified, 25 fi\. of PPD (100 fig. per ml.) were added by the incorporation of HEPES, a buffer to an equal volume of the above suspension, and for the control, physiologic phosphate Received October 21, 1974; received revised buffer replaced PPD solution. Both suspenmanuscript February 13, 1975; accepted for publisions were incubated at 37 C. for !4 hour, cation February 13, 1975. Address reprint requests to Dr. Petchclai. then each was placed in quadruplicate T H E L E U K O C Y T E MIGRATION T E S T
531
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
Koonakosit, Raungraiwan, and Petchclai, Bencha: Leukocyte migration test in a modified agarose medium. Am J Clin Pathol 64: 5 3 1 - 5 3 3 , 1975. The medium used for the leukocyte migration test contained 1% agarose, 0.003 M HEPES, TC medium 199, and 10% horse serum. T h e pH was adjusted to 7.3 with 2 N NaOH. T h e leukocyte migration test in this medium correlated with the intradermal test in 36 subjects when PPD was used as antigen, and the results were comparable to those of the reference technic. Addition of HEPES to the original medium enables the test to be performed in a standard incubator. (Key words: Migration test; Leukocyte; agarose medium.)
532
KOONAKOSIT AND PETCHCLAI Table 1. Mean, S.D., and Range of M.I. in 36 Subjects Range
Mean
S.D.
No.
Tuberculin pos.
0.74-0.39
.63
0.09
26
Tuberculin neg.
0.81-1.06
.94
0.07
10
A.J.C.P. —Vol. 64
Copenhagen) was dialyzed against physiologic saline solution at 4 C. to remove the preservatives. Results
Discussion where Mx = average areas of migration in test suspension; Mo = average areas of migration in control suspension. If 12.49 sq. mm. and 24.42 sq. mm. were average areas of migration of test and control suspensions, respectively, 12.49 M.I. = — — = 0.51. 24.42 Inhibition of migration in the presence of antigen will result in a smaller M.I., demonstrating cell-mediated immunity to that antigen. Results of the leukocyte migration test and skin test with PPD were compared in ten tuberculin-negative (from the pediatric ward) and 26 tuberculin-positive (adult) subjects. Mantoux test results were read 48 hours after intradermal injection of 5 T U of PPD (Parke Davis). PPD for the leukocyte migration test (Staten Serum Institute,
T h e modified medium is highly suitable for the leukocyte migration test. T h e incorporation of 0.003 M HEPES makes possible incubation in a standard incubator, which is more readily available, simpler, and less expensive to operate compared with a C 0 2 incubator. Migration areas in 36 subjects were in the range of 15.73-34.75 sq. mm., in agreement with the range of 8.75-59.56 sq. mm. found by Clausen. 5 Means of M.I. of the tuberculin-negative group (0.94) and the tuberculin-positive group (0.63) were very close to Clausen's corresponding values of 0.90 and 0.60. 5 T h e original work of Clausen 5 showed overlapping of M.I. in tuberculin-negative and tuberculin-positive groups, which was more pronounced when a higher strength of PPD was used for the intradermal test. 5 This suggests that the lack of overlapping M.I. in the
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
(7 fil.) into holes 2 mm. in diameter already cut into the agarose medium. T h e plastic agarose plates were put into a moist chamber and incubated at 37 C. in a standard incubator for 18-24 hours before measurement of migration and calculation of migration index (M.I.). T h e product of two diameters (of leukocyte migration) perpendicular to each other, measured by a magnifying micrometer, represents migration area. If a and b were such diameters, migration area = ab. Migration index (M.I.) can be calculated from the formula:
T h e color of the medium after incubation showed that its pH was well maintained. HEPES at a concentration of 0.003 M is the minimal amount maintaining the pH and permitting maximal migration of leukocytes. Migration areas of the 36 subjects had a mean of 23.12 sq. mm. (range 15.73-34.75 sq. mm.). Mean, range, and S.D. of M.I. in these subjects are shown in Table 1. T h e values in the tuberculin-negative and tuberculin-positive groups were significantly different (p < 0.001). There was no overlapping of M.I. between the two groups, and the values were clearly divided at the M.I. of 0.80. Correlations between M.I. and diameters of the cutaneous reactions in the tuberculin-positive group were highly significant (r = 0.8687, p < 0.001).
October 1975
LEUKOCYTE MIGRATION T E S T MEDIUM
533
Acknowledgment. Professor Natth Bhamarapravati, Chairman, Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, encouraged this study and edited the manuscript.
References 1. Anderson V, Bendixen G, Schi0dt T: An in vitro demonstration of cellular immunity against autologous mammary carcinoma in man. Acta Med Scand 186:101-103, 1969 2. Astor SH, Spitler LE, Frick OL, et al: Human leukocyte migration inhibition in agarose using four antigens: Correlation with skin reactivity. J Immunol 110:1174-1179, 1973 3. Brostoff J, Howell A, Roitt IM: Leukocyte migration inhibition with aggregated gamma globulin in patients with rheumatoid arthritis. Clin Exp Immunol 15:1-7, 1973 4. Clancy R: Cellular immunity to autologous
13. S0borg, M: In vitro demonstration of microbial cellular hypersensitivity in man. Proc R Soc Med 63:903-905, 1970 14. Thulin H, Zachariae H: T h e leukocyte migration test in chromium hypersensitivity. J Invest Dermatol 58:55-58, 1972 15. Valdimarsson H, Wood CBS, Hobbs JR, et al: Immunological features in a case of chronic granulomatous candidiasis and its treatment with transfer factor. Clin Exp Immunol 11:151-163, 1972 16. Wartenberg J, Doniach D, Brostoff J, et al: Leukocyte migration inhibition with mitochondria in human a u t o i m m u n e - t h y r o i d disorders. Clin Exp Immunol 14:203-212, 1973 17. Yanovsky JF, Albado E: Humoral and cellular response to Trypanosoma cruzi infection. J Immunol 109:1159-1161, 1972 18. Yeung Laiwah AAC, Chaudhuri AKR, Anderson JR: Lymphocyte transformation and leukocyte migration inhibition by Australia antigen. Clin Exp Immunol 15:27-34, 1973
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
platelets and serum blocking factors in idiotwo major groups in this study could be pathic thrombocytopenic purpura. Lancet 1: due, at least partly, to the use of only 5 T U 6 - 9 , 1972 for intradermal test. It is not known 5. Clausen JE: Tuberculin induced migration inhibition of human peripheral leukocytes in agarose whether age also plays a part, as the tubermedium. Acta Allerg 5 6 - 8 0 , 1971 culin-positive group was limited to adults. 6. Clausen J E : Migration inhibitory effect of cellfree supernatant from mixed human lymphoT h e excellent correlation between M.I. cyte cultures. J Immunol 108:453-459, 1972 and sizes of cutaneous reactions in the 7. Gaines J D , Araujo FG, Krahenbuhl JL, et al: tuberculin-positive g r o u p agreed with Simplified in vitro method for measuring delayed hypersensitivity to latent intracellular the findings of Clausen, 5 and proved once infection in man (toxoplasmosis). J Immunol again that the agarose technic for the 109:179-182, 1972 8. Galanaud P, Crevon MC, Dormont J, et al: leukocyte migration test could replace an Leukocyte migration test and renal allograft intradermal test, if necessary. rejection. Transplantation 13:48-52, 1972 T h e leukocyte migration test is used 9. Goldstone AH, Calder EA, Barnes EQ, et al: T h e effect of gastric antigens on the in vitro during immunotherapy 1 2 , 1 5 and tissue migration of leukocytes from patients with 8,11 to monitor cell-meditransplantation atrophic gastritis and pernicious anemia. Clin Exp Immunol 14:501-508, 1973 ated immunity to certain antigens. During 10. Kolt E, Genkins G, Rule AH: Leukocyte reimmunotherapy the leukocyte migration sponse to muscles antigens in myasthenia gravis. Relationship to clinical severity and test was somewhat better than the lymphopresence of circulating antibodies. Neurology cyte transformation test in monitoring the 23:374-380, 1973 12,15 T h e result is obtainable 11. Richmond DE, Doak PB, North JDK: Inhibition response. of leukocyte migration as an index of rejection within 24 hours. T h e test requires only in renal transplant recipients. Clin Exp 5 ml. of blood, and minimal reagents and Immunol 15:17-25, 1973 equipment. These advantages could lead to 12. Schulkind ML, Adler WH III, Altemeir WA: Transfer factor in the treatment of a case of adoption of this method by more clinical chronic mucocutaneous candidiasis. Cell laboratories. Immunol 3:606-615, 1972
M.D.
Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Bangkok 4, Thailand
ABSTRACT
(LMT) that increases the stability of the medium has been successfully used as an in-vitro in a standard incubator. It contained 1% test of cell-mediated immunity (CMI) to agarose (Sigma Chemical Co.,), 0.003 M microbial antigens, 2 " 7 , 1 3 , 1 7 , 1 8 autoanti- HEPES (Sigma Chemical Co.), TC medium gens, 3,4,9,10,16 chemical substances, 14 and 199 (Difco), and 10% horse serum. T h e pH even tumor antigens. 1 It is also used in was adjusted to 7.3 with 2 N NaOH and mixed lymphocyte c u l t u r e reactions then 15 ml. were poured into a 9-cm. (MLC) 8 and to detect transplant rejec- plastic Petri dish. tion. 8,11 T h e agarose plate method introThe collection of heparinized blood, duced by Clausen 5 is simple and highly dextran separation of leukocytes, and reproducible. It requires smaller amounts other technics followed the method of of leukocytes and antigens and less equip- Clausen 5 and are described here briefly. ment. T h e technic should prove more useFive milliliters of blood were collected ful for clinical application if it is further into a polyethylene syringe, then transsimplified. T h e present modified medium ferred into a siliconized screw-capped can be successfully used in an ordinary tube containing 125 I.U. heparin. After incubator. dextran sedimentation and washing, leukocytes were suspended in TC 199 Materials and Methods medium (containing 10% horse serum) to make a concentration of approximately T h e method followed that of Clausen, 5 2 x 105 cells per cu. mm. For the test, and only the medium was slightly modified, 25 fi\. of PPD (100 fig. per ml.) were added by the incorporation of HEPES, a buffer to an equal volume of the above suspension, and for the control, physiologic phosphate Received October 21, 1974; received revised buffer replaced PPD solution. Both suspenmanuscript February 13, 1975; accepted for publisions were incubated at 37 C. for !4 hour, cation February 13, 1975. Address reprint requests to Dr. Petchclai. then each was placed in quadruplicate T H E L E U K O C Y T E MIGRATION T E S T
531
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
Koonakosit, Raungraiwan, and Petchclai, Bencha: Leukocyte migration test in a modified agarose medium. Am J Clin Pathol 64: 5 3 1 - 5 3 3 , 1975. The medium used for the leukocyte migration test contained 1% agarose, 0.003 M HEPES, TC medium 199, and 10% horse serum. T h e pH was adjusted to 7.3 with 2 N NaOH. T h e leukocyte migration test in this medium correlated with the intradermal test in 36 subjects when PPD was used as antigen, and the results were comparable to those of the reference technic. Addition of HEPES to the original medium enables the test to be performed in a standard incubator. (Key words: Migration test; Leukocyte; agarose medium.)
532
KOONAKOSIT AND PETCHCLAI Table 1. Mean, S.D., and Range of M.I. in 36 Subjects Range
Mean
S.D.
No.
Tuberculin pos.
0.74-0.39
.63
0.09
26
Tuberculin neg.
0.81-1.06
.94
0.07
10
A.J.C.P. —Vol. 64
Copenhagen) was dialyzed against physiologic saline solution at 4 C. to remove the preservatives. Results
Discussion where Mx = average areas of migration in test suspension; Mo = average areas of migration in control suspension. If 12.49 sq. mm. and 24.42 sq. mm. were average areas of migration of test and control suspensions, respectively, 12.49 M.I. = — — = 0.51. 24.42 Inhibition of migration in the presence of antigen will result in a smaller M.I., demonstrating cell-mediated immunity to that antigen. Results of the leukocyte migration test and skin test with PPD were compared in ten tuberculin-negative (from the pediatric ward) and 26 tuberculin-positive (adult) subjects. Mantoux test results were read 48 hours after intradermal injection of 5 T U of PPD (Parke Davis). PPD for the leukocyte migration test (Staten Serum Institute,
T h e modified medium is highly suitable for the leukocyte migration test. T h e incorporation of 0.003 M HEPES makes possible incubation in a standard incubator, which is more readily available, simpler, and less expensive to operate compared with a C 0 2 incubator. Migration areas in 36 subjects were in the range of 15.73-34.75 sq. mm., in agreement with the range of 8.75-59.56 sq. mm. found by Clausen. 5 Means of M.I. of the tuberculin-negative group (0.94) and the tuberculin-positive group (0.63) were very close to Clausen's corresponding values of 0.90 and 0.60. 5 T h e original work of Clausen 5 showed overlapping of M.I. in tuberculin-negative and tuberculin-positive groups, which was more pronounced when a higher strength of PPD was used for the intradermal test. 5 This suggests that the lack of overlapping M.I. in the
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
(7 fil.) into holes 2 mm. in diameter already cut into the agarose medium. T h e plastic agarose plates were put into a moist chamber and incubated at 37 C. in a standard incubator for 18-24 hours before measurement of migration and calculation of migration index (M.I.). T h e product of two diameters (of leukocyte migration) perpendicular to each other, measured by a magnifying micrometer, represents migration area. If a and b were such diameters, migration area = ab. Migration index (M.I.) can be calculated from the formula:
T h e color of the medium after incubation showed that its pH was well maintained. HEPES at a concentration of 0.003 M is the minimal amount maintaining the pH and permitting maximal migration of leukocytes. Migration areas of the 36 subjects had a mean of 23.12 sq. mm. (range 15.73-34.75 sq. mm.). Mean, range, and S.D. of M.I. in these subjects are shown in Table 1. T h e values in the tuberculin-negative and tuberculin-positive groups were significantly different (p < 0.001). There was no overlapping of M.I. between the two groups, and the values were clearly divided at the M.I. of 0.80. Correlations between M.I. and diameters of the cutaneous reactions in the tuberculin-positive group were highly significant (r = 0.8687, p < 0.001).
October 1975
LEUKOCYTE MIGRATION T E S T MEDIUM
533
Acknowledgment. Professor Natth Bhamarapravati, Chairman, Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, encouraged this study and edited the manuscript.
References 1. Anderson V, Bendixen G, Schi0dt T: An in vitro demonstration of cellular immunity against autologous mammary carcinoma in man. Acta Med Scand 186:101-103, 1969 2. Astor SH, Spitler LE, Frick OL, et al: Human leukocyte migration inhibition in agarose using four antigens: Correlation with skin reactivity. J Immunol 110:1174-1179, 1973 3. Brostoff J, Howell A, Roitt IM: Leukocyte migration inhibition with aggregated gamma globulin in patients with rheumatoid arthritis. Clin Exp Immunol 15:1-7, 1973 4. Clancy R: Cellular immunity to autologous
13. S0borg, M: In vitro demonstration of microbial cellular hypersensitivity in man. Proc R Soc Med 63:903-905, 1970 14. Thulin H, Zachariae H: T h e leukocyte migration test in chromium hypersensitivity. J Invest Dermatol 58:55-58, 1972 15. Valdimarsson H, Wood CBS, Hobbs JR, et al: Immunological features in a case of chronic granulomatous candidiasis and its treatment with transfer factor. Clin Exp Immunol 11:151-163, 1972 16. Wartenberg J, Doniach D, Brostoff J, et al: Leukocyte migration inhibition with mitochondria in human a u t o i m m u n e - t h y r o i d disorders. Clin Exp Immunol 14:203-212, 1973 17. Yanovsky JF, Albado E: Humoral and cellular response to Trypanosoma cruzi infection. J Immunol 109:1159-1161, 1972 18. Yeung Laiwah AAC, Chaudhuri AKR, Anderson JR: Lymphocyte transformation and leukocyte migration inhibition by Australia antigen. Clin Exp Immunol 15:27-34, 1973
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
platelets and serum blocking factors in idiotwo major groups in this study could be pathic thrombocytopenic purpura. Lancet 1: due, at least partly, to the use of only 5 T U 6 - 9 , 1972 for intradermal test. It is not known 5. Clausen JE: Tuberculin induced migration inhibition of human peripheral leukocytes in agarose whether age also plays a part, as the tubermedium. Acta Allerg 5 6 - 8 0 , 1971 culin-positive group was limited to adults. 6. Clausen J E : Migration inhibitory effect of cellfree supernatant from mixed human lymphoT h e excellent correlation between M.I. cyte cultures. J Immunol 108:453-459, 1972 and sizes of cutaneous reactions in the 7. Gaines J D , Araujo FG, Krahenbuhl JL, et al: tuberculin-positive g r o u p agreed with Simplified in vitro method for measuring delayed hypersensitivity to latent intracellular the findings of Clausen, 5 and proved once infection in man (toxoplasmosis). J Immunol again that the agarose technic for the 109:179-182, 1972 8. Galanaud P, Crevon MC, Dormont J, et al: leukocyte migration test could replace an Leukocyte migration test and renal allograft intradermal test, if necessary. rejection. Transplantation 13:48-52, 1972 T h e leukocyte migration test is used 9. Goldstone AH, Calder EA, Barnes EQ, et al: T h e effect of gastric antigens on the in vitro during immunotherapy 1 2 , 1 5 and tissue migration of leukocytes from patients with 8,11 to monitor cell-meditransplantation atrophic gastritis and pernicious anemia. Clin Exp Immunol 14:501-508, 1973 ated immunity to certain antigens. During 10. Kolt E, Genkins G, Rule AH: Leukocyte reimmunotherapy the leukocyte migration sponse to muscles antigens in myasthenia gravis. Relationship to clinical severity and test was somewhat better than the lymphopresence of circulating antibodies. Neurology cyte transformation test in monitoring the 23:374-380, 1973 12,15 T h e result is obtainable 11. Richmond DE, Doak PB, North JDK: Inhibition response. of leukocyte migration as an index of rejection within 24 hours. T h e test requires only in renal transplant recipients. Clin Exp 5 ml. of blood, and minimal reagents and Immunol 15:17-25, 1973 equipment. These advantages could lead to 12. Schulkind ML, Adler WH III, Altemeir WA: Transfer factor in the treatment of a case of adoption of this method by more clinical chronic mucocutaneous candidiasis. Cell laboratories. Immunol 3:606-615, 1972
