Journal of Immunological Methods, 16 (1977) 269--281
26c~
© Elsevier/North-Holland Biomedical Press
A MICROASSAY FOR LEUKOCYTE MIGRATION: ANALYSIS OF ITS REPRODUCIBILITY
RICHARD H. WEISBART avd M. RAY MICKEY Department of Medicine and Department of Biomathematics, UCLA Center for the Health Sciences, Los Angeles, California 90024. U.S.A.
(Received 15 November 1976, accepted 1~3February 1977)
A reliable microassay for human leukocyte migration is described by which 50 to 100 assays can be performed each day in qua~truplicate with the number of indicator cells obtainable from 10 to 20 ml of peril~heral blood. The reproducibility of this method is demonstrated with respect to the variability among replicate test wells (including reading), the variability among different test readers, the variability among replicate cultures, and the variability of using indicator cells from different subjects. A microculture system is described that requires only 75,000 mononuclear cells to consistently produce detectable polymorphonuclear leukocyte migration inhibition factor in response to PPD. The reproducibility of this culture system is demonstrated with respect to the variability of lymphocyte responsiveness on repetitive testing in the same individuals.
INTRODUCTION Soluble factors ~'e produced by sensitized l y m p h o c y t e s as a result of spe~fic antigen stimulation. These substances are t h o u g h t to mediate mechanisms of l y m p h o c y t e reactivity involved in the defense against infectious agents, allograft rejection, surveillance against neoplasia, and the pathogenesis o f certain a u t o i m m u n e diseases. Among the most extensively studied mediators are those that alter the migration of phagocytic cells. Migration assays have limited clinical usefulness, however, because o f the technicai complexity of performing relatively few tests. Recent attempts to resolve these limitations include utilizing polymori~honuclear leukocytes as indlicator cells, migrating cells under an agar gel, and miniaturizing test methods (Carpenter et al., 1968; Clausen, 1971; Harrington et al., 1973). Even with these improvements, reproducibility remains the ma!or point o f c o n t e n t i o n in migration asmys. In this report we describe a microassay for l e u k o c y t e migration and assess its reproducibility with respect to the variability among replicate test wells (including reading), the variability among different test readers, the variability among replicate cultures, and the variability o f using indicator cells from different subjects. We also determined the n u m b e r o f cells required to produce detectable mediator in response t o antigenic stimulation. Since reproducible stimulation of mediator production is
270 necessary for meaningful clinical application, we ascertained the variability of lymphocyte responsiveness to antigen on repetitive testing in the same individuals. MATERIALS
AND METHODS
Subjects studied Peripheral blood was obtained from 16 healthy subjects.
Antigen Tween stabilized purified protein derivative (PPD) (Connaught Laboratories Limited, Ontario, Canada) was used to test for delayed intracutaneous skin reactivity. Skin testing was done after the in vitro studies were completed. A positive reaction was defined as an area of induration equal to or greater than 10 m m . Subjects unresponsive to the intermediate dose of 5 tuberculin units were subsequently skin tested with the second strength dose (250 tuberculin units). PPD (lot 974775) used for lymphocyte cultures was donated by H.B. Delvin, PhoD., Parke-Davis Company.
Lymphocyte separation and culture technique Lymphocytes were isolated from peripheral blood by gravity sedimentation as previously described except that autologous plasma was used instead of serum (Weisbart et al., 1972}. The cells were washed 3 times with Hank's Buffered Salt Solution (HBSS) and cultured in 12 X 75 m m pclypropylene tubes (Falcon Plastics, Oxnard, California) in medium 199 witb 25 mM Hepes buffer, 2.2 g NaHCO3 per liter (Grand Island Biological Company, Berkeley, California), 10% horse serum, penicillin 100 units per ml and streptomycin 100/~g per ml. Cultures were done in pairs, with an antigen stimulated culture ar.~ a non-stimulated culture to which a comparable volume of HBSS wa~" added. The cultures were incubated at 37°C in the presence of 5% CO2. Aiiquots of each lymphocyte culture supernatant were removed at various periods of time between one and seven days mid stored at --20°C in Micro Test II plates sealed with pressure-sensitive film (Falcon Plastics, Oxnard, California). The non-stimulated supernatants were reconstituted with PPD, and an equivalent volume of HBSS was added to each stimulated culture supematant. At the termination of the cultures, the tubes were centrifuged for 10 rain at 2000 g mid the remaining supernatant was decanted. Eighty percent or more of the cells present at the end of the culture period were viable as determined by trypan blue exclusion.
Agarose gel techniqe Preparation of agarose ~nedium A 2% solution of agarose in m e d i u m 199 was prepaied as previously de-
271 scribed (Weisbart et al., ~ 9~13), and adjusted to contain ~0% horse serum and 1% agarose, penicillin (] 00 units/ml}, and streptomycin (100/~g/ml). In Experiment A, 2 ml of agar ~veredispended in 35 mm round t}.ssue cultur~ dishes (Falcon Plastics, Oxrlard, Calfomia). Eight wells of 1.5 mm diameter were cut in a circular pattern in each dish, and the agar plugs were gently aspirated immediately before dispensing migrating cells into the wells. Groups of four plates were contained in covered 100 mm square petri dishes with moistened cotton to prevent drying of the agar medium. In experiments B and C, 30 ml of agar were dispensed in 150 mm round tissue culture dishes (integrid, Falcon Plastics, Oxnard, California}. One hundred twenty-eight wells of 1.5 mm diameter were cut in each plate. The b~tegrid dish contains 32 squares; four wells were cut in each square, and 2 squares consisting of two rows of 4 wells each were used for each test. The agar plugs were gently aspirated, and the plates were placed in a humidified incubator at 37°C for 5--10 rain until the emptied wells became partially filled with liquid from the agar medium. At this time, the surface of the agar was covered with 15 ml of light-weight mineral oil (Lubeno!, Purepac Corp., Elizabeth, New Jersey}. The plates were incubatecl at 37°C with 5% CO2 until the wells were ready to be filled with migrating cells.
Preparation of leukocyte indicator cells In Experiment A buffy coat cells were obtained by sedimentmg red blood cells with dextran (9 parts Dextran-250 to 25 parts of blood} fl~r 1 h. It was found necessary to maintain the red blood cell to white blood cell ratio at less than 10 : 1. The buffy coat cells were washed three times with 2% horse serum in HBSS. In Experiments B and C, purified polymorphonuclear leukocytes (97--99%) were isolated from peripheral blood by Ficoll--Hypaque gradient centrifugation (Boyum, 1968,). The red blood cell layer was recovered from the gradients and diluted with an equal volume o f HBSS. The red blood cells were then removed by dextran sedimentation so that the red cell to polymorphonuclear leukocyte ratio was less than 10 : 1; the average ratio was about 3 : 1. The cells were washed three times witb 2% horse serum in HBSS.
Agarose technique Buffy coat cells were resuspended to contain 50 × 106 polymorphonuclear leukocytes/ml in medium 199 with 25 mM Hepes buffer and 10% horse serum. Four pl aliquots of the cell suspension were dispensed (microdispenser, Drummond Scientific Company, Broomal, Pennsylvania) into each well of a micro test tissue culture plate (Falcon Plastics, Oxnard, California). Four/~1 aliquots of each lymphocyte cultvre supernatant were added to and mixed With each cell suspension with the microdispenser. Evaporation was prevented by covering each microtiter well with a drop of light-weight mineral oil. The micro test plates were ivcubated for 30 rain at 37°C. The contents of each microtiter well were arawn into a 50 pl syringe (14 inch
272 long, 0.0045 inch I.D. replaceable needle) attached to an automatic dispenser (Unimetrics Corporation, Anaheim, California), mixed, and 1 ml aliquots were dispensed into each of four wells in an agarose dish. In Experiment A, the cells were distributed according to 24 different patterns marked on plastic cards and visualized through the agar dish. This variation of pattern distribution permitted blind assessment of the tests. In Experiments B and C, 1 pl aliquots of each cell supernatant suspension were dispended into each of four wells in an alternating, pattern between two rows of wells. The agar plates were incubated for 6 h at 37°C in a humidified incubator with 95% room air and 5% CO2. The dishes were then filled with 95% ethyl alcohol for 6 or more hours, after which time, the agar was gently lifted from the dishes. The dishes dried quickly without spotting and provided a permanent prepar=~tion for reading. In Experiment A, the order of the plates was selected for reading by the use of random number tables and presented to the reader for blind assessment. In Experiments B and C, the tests were distributed horizontally in each plate so that unbiased reading was achieved by measuring the migrations in vertical rows; only the migration being measured could be visualized. Fig. 3. shows an example of 16 quadruplicate tests in the 150 mm tissue c'J!ture dish.
Data analysis The largest diameter of each migration was measured with an ocular micrometer. The main analysis was constructed from the differences between the average control migration diameter and the average test diameter (control minus tesL). Analysis of variance calculations (Snedecor et al., 1967) were computed using programs BMDO8V and BMDIOV (Dixon, 1973).
Experimental Design A This experiment was designe~t to provide information about the effects of antigen concentration in lymphocyte cultures and the duration of culture on the migration of indicator cells incubated with aliquots of culture supernatant. The experiment was planned to give infnnnation about the variability among replicate test wells (including reading), the variability of replicate cultures, and the variability of u~ing indicator cells from different subjects. Two tubes of blood were obtained from each of 16 sabjects and lymphocytes were independently isolated from each tube. One million lymphocytes from each tube were cultured in 1 ml of medium with two concentrations of PPD, 25 and 6.25/~g/ml. Thirty gl of supernatant were removed from each culture after 1, 2, 3~ 5, 6, and 7 day(s) of culture. Control cul'ures were done in parallel witb the test cultures. The experimental design arranged the work to be done in eight assay days alternating the use of indicator cells from two individuals (table 1). The cultures from eight subjects were tested on each day with each of the 6 durations of culture tested at the same time.
273
Fig. 1. Migration of potymorphonucleac ieukocytes. Each culture is assayed in quadruplicate and consists of eight, migrations occupying two horizontally adjacent squares. Indicator cells incubated with control and stimulated lymphocyte supernatants are alternated in two horizontal rows beginning with the control migration in the upper left. Each assay then consisted of 8 (donors) X 6 (culture durations) X 4 (quadruplicate) X 2 (test/control) = 384 wells tested in 48 plates.
Experimental Design B This experiment was designed to s t u d y the effect o f n u m b e r of cells in culture and the concentration o f antigen on the migration o f indicator cells incubated with culture supernatant. Cultures were established in 0.5 ml volumes at t:hree cell concentrations. In 5 subjects the cell concentrations were 4.0 X 106/ml, 1.0 X 106/ml, and 0.25 X 10°/mi. Limited cell numbers were obtained in the remaining 4 subjects necessitating use o f the following
TABLE 1
1 2
1 2
1 2
1 2
1
2
3
4
1 2
1 2
1 2
1 2
PMN donor
Ab
Ba
Bb
Aa
1
Ba
Ab
Aa
Bb
2
Bb
Aa
Ab
Ba
3
Aa
Bb
Ba
Ab
4
L y m p h o c y t e don o r 5
Ab
Ba
Bb
Aa
6
Aa
Ab
Ba
Bb
Bb
Aa
Ab
Ba
7
Ba
Bb
Aa
Ab
8
9
Ab
Ba
Bb
Aa
10
Ba
Ab
Aa
Bb
Aa
Bb
Ab
Ba
11
Bb
Aa
Ba
Ab
12
13
Ab
Ba
Bb
Aa
14
Ba
Aa
Ab
Bb
Bb
Ab
Aa
Ba
15
Aa
Bb
Ba
Ab
16
A = 1st duplicate culture; B = 2nd duplicate culture; a = PPD, 2 5 / l g / m l ; b = PPD, 6.25 pg/mi. Each letter pair designated a set of 6 time p e r ~ d tests.
Day
Week
Test plan, experiment A.
275 concentrations: 2.5 X 10~/ml, 0.63 X 106/ml, and 0.16 X 106/ml. For each subject the 3 concentrations of PPD were 25 ~g/ml, 5 pg/ml, and 1 pg/ml. Nine donors were tested and cultures of each of the nine combinations of cell numbers and antige~ concentrations were made. The supematant was harvested on the ,lth day of culture. Control cultures without antigen were prepared at th,~. same time as the test cultures. The (test/control) 2 X 81 = 162 cultures were assayed at one time with migrating cells of a single donor, and quadruplic~te migration wells were prepared for each culture, with each test control pair tested on the same plate. These data were used to test the consistency of different readers. E~ch of 3 readers measured the diameter of each of the 648 migrations (to the nev:~'est 0.5 mm). In addition, these data were used to assess the degree of lymphocyte responsive[~ess to PPD in different individuals for the purpose of correlating the respm~se in vitro with the delayed hypersensitivity skin reaction.
Experimental De.~.ign C This experiment was designed to study the effects of the number of cultured lymphocytes and culture volume on the migration of indicator cells incubated with aliquots of the culture supernatants; in addition, we assessed the variability of lymphocyte responsiveness to antigen in the same subjects tested at different periods of time. Four replicate culture periods were established at 3 to 4 day intervals. At each period, lymphocytes from the same six donors were cultured with 25/~g/ml of PPD with all 9 combinations of cell concentrations of 0.5 × 106/ml, 0.1 X 106/ml, and 0.02 × 106/ml with culture volumes of 1 ml (16 X 125 mm culture tubes), 0.5 ml (12 × 75 mm culture tubes), and 0.150 ml (microtest tissue culture plates). Lymphocyte supematants were harvested on the 4th day of culture. Migrating cells from a single dohor were used, and quadruplicate wells of t~;st and control supernatants from one culture period were tested at the same time. RESULTS
Experiment A The results of experiment A given in table 2 are average values, the average being taken over all other factors. Of the 16 subjects, one was non-reactive, two gave weak reactions, and the other 13 had a fairly uniform strong response. The responses of subjects 13 and 4 were significantly different at P = 0.05, but not at P = 0.01 (multiple range test; Snedecor et al., 1967).
Effect o f antigen concentration on the migration o f indicator cells incubated with culture supernatant Antigen concentration was an important determinant of the response observed, for the average differential migration was 1.06 mm at 25 #g/ml of
276 TABLE 2 Average differential migration. Subject No.
Average migration difference (mm) con troi-test
Skin test response to PPD Intermediate
2nd strength
"6 1 12 13 2 5 8 16
0.151 0.547 0.547 0.870 0.922 0.943 0.943 0.984
-+ , + + ----
+
11
1.000
--
--
15
1.005
--
+
1.026
--
+
10
1.026
--
--
9
1.036
+
14
3
+ ND + --
1.089
--
+
7
1.182
--
--
4
1.229
ND
ND = not done. Standard error of the average differential migration = 0.080 (15 d.f.).
PPD and 0.752 m m at 6.25 pg/ml (the standard error o f the differential concentration mean was 0.047, 15 d.f., P < 0.001 for difference between average differential migrations).
Effect of duration of culture on the migration of indicator cells incubated with culture supernatant Statistically there was a strong effect of culture time on m e d i a t o r activity. The results are given in fig. 2. There was a small b u t definite increase in the migration diameter in the control wells and a decrease in the test wells, although the difference between contr,~! and test was very well established after one day. These data were used to compare expressing the d a t a as mean differential migration diameters with the more conventional use o f percent inhibition in the area of migration. As shown in fig. 2, there is n o difference between these two m e t h o d s o f expressing the data.
Reproducibility of the microassay for leukocyte migration The data were quite consistent. The mean square o f all interaction terms with days (from a subject (16) × concentration (2) X replicates (2) X days (6) analysis o f variance, which ignores some aspects o f the design) had t h e value o f 0.0615 m m 2 (315 d.f.). The largest term 0.0968 (75 d.f.) was the interaction of subjects with days, which would be expected to be s o m e w h a t larger because o f the non-responsiveness o f a few subjects. The value o f the 4-
277
CONTROL
TEST •
•
i"
6-
CONTROL-TEST
2O io I 1
I 2
I 3
I 4
I 5
I 6
I
7
DAYS OF CULTURE
Fig. 2. Effect of duration of culture on the migration of indicator cells incubated with culture supernatant. factor interaction, 0.0480 (75 d.f.), was consistent with what would be expected from quadruplicate tests, 0.037 (2304 d.f.). (The mean square of 768 se~s of quadruplicate wells was 0.0740. The variance of the difference b e t w e e n two averages of 4 wells each was estimated as 1/2 the pooled mean square, 0.0740/2 = 0.0370.) The effect of replicate cultures was estimated from the pooled interaction o f replicate cultures with subjects, concentration, and subject X concentration (31 d.f.). The effect was estimated as equivalent to adding a random effect with standard deviation of 0.2 rnm. No significant difference could be ascribed to using migrating cells from two different individuals on the basis of a non-orthogGnal analysis of variance c o m p u t e d using program BMD1OV (Dixon, 1973).
Experiment B Effect o f antigen and cell concentration on the migration: o f indicator cells incubated with culture supernatent The differences between average control and test migration diameter was c o m p u t e d for each reader-culture combination. The average difference for the factors of interest are p l o t t e J in fig. 3. The results indicate that even quite small numbers of cells are effective ~:jven adequate concentrations o f antigen. What is n o t shown b y the figure is :,hat the cultures were n o t consis-
278 ..=.
~
1.0 4x
~
.8
~
.6
~
.4
......
.5 x 106cells/ml
1.0-
106cells/ml
l x 106 cells/ml
1.2 -
~
. ~ ...---" ~sx lO°c~,,~m, I
26c~
© Elsevier/North-Holland Biomedical Press
A MICROASSAY FOR LEUKOCYTE MIGRATION: ANALYSIS OF ITS REPRODUCIBILITY
RICHARD H. WEISBART avd M. RAY MICKEY Department of Medicine and Department of Biomathematics, UCLA Center for the Health Sciences, Los Angeles, California 90024. U.S.A.
(Received 15 November 1976, accepted 1~3February 1977)
A reliable microassay for human leukocyte migration is described by which 50 to 100 assays can be performed each day in qua~truplicate with the number of indicator cells obtainable from 10 to 20 ml of peril~heral blood. The reproducibility of this method is demonstrated with respect to the variability among replicate test wells (including reading), the variability among different test readers, the variability among replicate cultures, and the variability of using indicator cells from different subjects. A microculture system is described that requires only 75,000 mononuclear cells to consistently produce detectable polymorphonuclear leukocyte migration inhibition factor in response to PPD. The reproducibility of this culture system is demonstrated with respect to the variability of lymphocyte responsiveness on repetitive testing in the same individuals.
INTRODUCTION Soluble factors ~'e produced by sensitized l y m p h o c y t e s as a result of spe~fic antigen stimulation. These substances are t h o u g h t to mediate mechanisms of l y m p h o c y t e reactivity involved in the defense against infectious agents, allograft rejection, surveillance against neoplasia, and the pathogenesis o f certain a u t o i m m u n e diseases. Among the most extensively studied mediators are those that alter the migration of phagocytic cells. Migration assays have limited clinical usefulness, however, because o f the technicai complexity of performing relatively few tests. Recent attempts to resolve these limitations include utilizing polymori~honuclear leukocytes as indlicator cells, migrating cells under an agar gel, and miniaturizing test methods (Carpenter et al., 1968; Clausen, 1971; Harrington et al., 1973). Even with these improvements, reproducibility remains the ma!or point o f c o n t e n t i o n in migration asmys. In this report we describe a microassay for l e u k o c y t e migration and assess its reproducibility with respect to the variability among replicate test wells (including reading), the variability among different test readers, the variability among replicate cultures, and the variability o f using indicator cells from different subjects. We also determined the n u m b e r o f cells required to produce detectable mediator in response t o antigenic stimulation. Since reproducible stimulation of mediator production is
270 necessary for meaningful clinical application, we ascertained the variability of lymphocyte responsiveness to antigen on repetitive testing in the same individuals. MATERIALS
AND METHODS
Subjects studied Peripheral blood was obtained from 16 healthy subjects.
Antigen Tween stabilized purified protein derivative (PPD) (Connaught Laboratories Limited, Ontario, Canada) was used to test for delayed intracutaneous skin reactivity. Skin testing was done after the in vitro studies were completed. A positive reaction was defined as an area of induration equal to or greater than 10 m m . Subjects unresponsive to the intermediate dose of 5 tuberculin units were subsequently skin tested with the second strength dose (250 tuberculin units). PPD (lot 974775) used for lymphocyte cultures was donated by H.B. Delvin, PhoD., Parke-Davis Company.
Lymphocyte separation and culture technique Lymphocytes were isolated from peripheral blood by gravity sedimentation as previously described except that autologous plasma was used instead of serum (Weisbart et al., 1972}. The cells were washed 3 times with Hank's Buffered Salt Solution (HBSS) and cultured in 12 X 75 m m pclypropylene tubes (Falcon Plastics, Oxnard, California) in medium 199 witb 25 mM Hepes buffer, 2.2 g NaHCO3 per liter (Grand Island Biological Company, Berkeley, California), 10% horse serum, penicillin 100 units per ml and streptomycin 100/~g per ml. Cultures were done in pairs, with an antigen stimulated culture ar.~ a non-stimulated culture to which a comparable volume of HBSS wa~" added. The cultures were incubated at 37°C in the presence of 5% CO2. Aiiquots of each lymphocyte culture supernatant were removed at various periods of time between one and seven days mid stored at --20°C in Micro Test II plates sealed with pressure-sensitive film (Falcon Plastics, Oxnard, California). The non-stimulated supernatants were reconstituted with PPD, and an equivalent volume of HBSS was added to each stimulated culture supematant. At the termination of the cultures, the tubes were centrifuged for 10 rain at 2000 g mid the remaining supernatant was decanted. Eighty percent or more of the cells present at the end of the culture period were viable as determined by trypan blue exclusion.
Agarose gel techniqe Preparation of agarose ~nedium A 2% solution of agarose in m e d i u m 199 was prepaied as previously de-
271 scribed (Weisbart et al., ~ 9~13), and adjusted to contain ~0% horse serum and 1% agarose, penicillin (] 00 units/ml}, and streptomycin (100/~g/ml). In Experiment A, 2 ml of agar ~veredispended in 35 mm round t}.ssue cultur~ dishes (Falcon Plastics, Oxrlard, Calfomia). Eight wells of 1.5 mm diameter were cut in a circular pattern in each dish, and the agar plugs were gently aspirated immediately before dispensing migrating cells into the wells. Groups of four plates were contained in covered 100 mm square petri dishes with moistened cotton to prevent drying of the agar medium. In experiments B and C, 30 ml of agar were dispensed in 150 mm round tissue culture dishes (integrid, Falcon Plastics, Oxnard, California}. One hundred twenty-eight wells of 1.5 mm diameter were cut in each plate. The b~tegrid dish contains 32 squares; four wells were cut in each square, and 2 squares consisting of two rows of 4 wells each were used for each test. The agar plugs were gently aspirated, and the plates were placed in a humidified incubator at 37°C for 5--10 rain until the emptied wells became partially filled with liquid from the agar medium. At this time, the surface of the agar was covered with 15 ml of light-weight mineral oil (Lubeno!, Purepac Corp., Elizabeth, New Jersey}. The plates were incubatecl at 37°C with 5% CO2 until the wells were ready to be filled with migrating cells.
Preparation of leukocyte indicator cells In Experiment A buffy coat cells were obtained by sedimentmg red blood cells with dextran (9 parts Dextran-250 to 25 parts of blood} fl~r 1 h. It was found necessary to maintain the red blood cell to white blood cell ratio at less than 10 : 1. The buffy coat cells were washed three times with 2% horse serum in HBSS. In Experiments B and C, purified polymorphonuclear leukocytes (97--99%) were isolated from peripheral blood by Ficoll--Hypaque gradient centrifugation (Boyum, 1968,). The red blood cell layer was recovered from the gradients and diluted with an equal volume o f HBSS. The red blood cells were then removed by dextran sedimentation so that the red cell to polymorphonuclear leukocyte ratio was less than 10 : 1; the average ratio was about 3 : 1. The cells were washed three times witb 2% horse serum in HBSS.
Agarose technique Buffy coat cells were resuspended to contain 50 × 106 polymorphonuclear leukocytes/ml in medium 199 with 25 mM Hepes buffer and 10% horse serum. Four pl aliquots of the cell suspension were dispensed (microdispenser, Drummond Scientific Company, Broomal, Pennsylvania) into each well of a micro test tissue culture plate (Falcon Plastics, Oxnard, California). Four/~1 aliquots of each lymphocyte cultvre supernatant were added to and mixed With each cell suspension with the microdispenser. Evaporation was prevented by covering each microtiter well with a drop of light-weight mineral oil. The micro test plates were ivcubated for 30 rain at 37°C. The contents of each microtiter well were arawn into a 50 pl syringe (14 inch
272 long, 0.0045 inch I.D. replaceable needle) attached to an automatic dispenser (Unimetrics Corporation, Anaheim, California), mixed, and 1 ml aliquots were dispensed into each of four wells in an agarose dish. In Experiment A, the cells were distributed according to 24 different patterns marked on plastic cards and visualized through the agar dish. This variation of pattern distribution permitted blind assessment of the tests. In Experiments B and C, 1 pl aliquots of each cell supernatant suspension were dispended into each of four wells in an alternating, pattern between two rows of wells. The agar plates were incubated for 6 h at 37°C in a humidified incubator with 95% room air and 5% CO2. The dishes were then filled with 95% ethyl alcohol for 6 or more hours, after which time, the agar was gently lifted from the dishes. The dishes dried quickly without spotting and provided a permanent prepar=~tion for reading. In Experiment A, the order of the plates was selected for reading by the use of random number tables and presented to the reader for blind assessment. In Experiments B and C, the tests were distributed horizontally in each plate so that unbiased reading was achieved by measuring the migrations in vertical rows; only the migration being measured could be visualized. Fig. 3. shows an example of 16 quadruplicate tests in the 150 mm tissue c'J!ture dish.
Data analysis The largest diameter of each migration was measured with an ocular micrometer. The main analysis was constructed from the differences between the average control migration diameter and the average test diameter (control minus tesL). Analysis of variance calculations (Snedecor et al., 1967) were computed using programs BMDO8V and BMDIOV (Dixon, 1973).
Experimental Design A This experiment was designe~t to provide information about the effects of antigen concentration in lymphocyte cultures and the duration of culture on the migration of indicator cells incubated with aliquots of culture supernatant. The experiment was planned to give infnnnation about the variability among replicate test wells (including reading), the variability of replicate cultures, and the variability of u~ing indicator cells from different subjects. Two tubes of blood were obtained from each of 16 sabjects and lymphocytes were independently isolated from each tube. One million lymphocytes from each tube were cultured in 1 ml of medium with two concentrations of PPD, 25 and 6.25/~g/ml. Thirty gl of supernatant were removed from each culture after 1, 2, 3~ 5, 6, and 7 day(s) of culture. Control cul'ures were done in parallel witb the test cultures. The experimental design arranged the work to be done in eight assay days alternating the use of indicator cells from two individuals (table 1). The cultures from eight subjects were tested on each day with each of the 6 durations of culture tested at the same time.
273
Fig. 1. Migration of potymorphonucleac ieukocytes. Each culture is assayed in quadruplicate and consists of eight, migrations occupying two horizontally adjacent squares. Indicator cells incubated with control and stimulated lymphocyte supernatants are alternated in two horizontal rows beginning with the control migration in the upper left. Each assay then consisted of 8 (donors) X 6 (culture durations) X 4 (quadruplicate) X 2 (test/control) = 384 wells tested in 48 plates.
Experimental Design B This experiment was designed to s t u d y the effect o f n u m b e r of cells in culture and the concentration o f antigen on the migration o f indicator cells incubated with culture supernatant. Cultures were established in 0.5 ml volumes at t:hree cell concentrations. In 5 subjects the cell concentrations were 4.0 X 106/ml, 1.0 X 106/ml, and 0.25 X 10°/mi. Limited cell numbers were obtained in the remaining 4 subjects necessitating use o f the following
TABLE 1
1 2
1 2
1 2
1 2
1
2
3
4
1 2
1 2
1 2
1 2
PMN donor
Ab
Ba
Bb
Aa
1
Ba
Ab
Aa
Bb
2
Bb
Aa
Ab
Ba
3
Aa
Bb
Ba
Ab
4
L y m p h o c y t e don o r 5
Ab
Ba
Bb
Aa
6
Aa
Ab
Ba
Bb
Bb
Aa
Ab
Ba
7
Ba
Bb
Aa
Ab
8
9
Ab
Ba
Bb
Aa
10
Ba
Ab
Aa
Bb
Aa
Bb
Ab
Ba
11
Bb
Aa
Ba
Ab
12
13
Ab
Ba
Bb
Aa
14
Ba
Aa
Ab
Bb
Bb
Ab
Aa
Ba
15
Aa
Bb
Ba
Ab
16
A = 1st duplicate culture; B = 2nd duplicate culture; a = PPD, 2 5 / l g / m l ; b = PPD, 6.25 pg/mi. Each letter pair designated a set of 6 time p e r ~ d tests.
Day
Week
Test plan, experiment A.
275 concentrations: 2.5 X 10~/ml, 0.63 X 106/ml, and 0.16 X 106/ml. For each subject the 3 concentrations of PPD were 25 ~g/ml, 5 pg/ml, and 1 pg/ml. Nine donors were tested and cultures of each of the nine combinations of cell numbers and antige~ concentrations were made. The supematant was harvested on the ,lth day of culture. Control cultures without antigen were prepared at th,~. same time as the test cultures. The (test/control) 2 X 81 = 162 cultures were assayed at one time with migrating cells of a single donor, and quadruplic~te migration wells were prepared for each culture, with each test control pair tested on the same plate. These data were used to test the consistency of different readers. E~ch of 3 readers measured the diameter of each of the 648 migrations (to the nev:~'est 0.5 mm). In addition, these data were used to assess the degree of lymphocyte responsive[~ess to PPD in different individuals for the purpose of correlating the respm~se in vitro with the delayed hypersensitivity skin reaction.
Experimental De.~.ign C This experiment was designed to study the effects of the number of cultured lymphocytes and culture volume on the migration of indicator cells incubated with aliquots of the culture supernatants; in addition, we assessed the variability of lymphocyte responsiveness to antigen in the same subjects tested at different periods of time. Four replicate culture periods were established at 3 to 4 day intervals. At each period, lymphocytes from the same six donors were cultured with 25/~g/ml of PPD with all 9 combinations of cell concentrations of 0.5 × 106/ml, 0.1 X 106/ml, and 0.02 × 106/ml with culture volumes of 1 ml (16 X 125 mm culture tubes), 0.5 ml (12 × 75 mm culture tubes), and 0.150 ml (microtest tissue culture plates). Lymphocyte supematants were harvested on the 4th day of culture. Migrating cells from a single dohor were used, and quadruplicate wells of t~;st and control supernatants from one culture period were tested at the same time. RESULTS
Experiment A The results of experiment A given in table 2 are average values, the average being taken over all other factors. Of the 16 subjects, one was non-reactive, two gave weak reactions, and the other 13 had a fairly uniform strong response. The responses of subjects 13 and 4 were significantly different at P = 0.05, but not at P = 0.01 (multiple range test; Snedecor et al., 1967).
Effect o f antigen concentration on the migration o f indicator cells incubated with culture supernatant Antigen concentration was an important determinant of the response observed, for the average differential migration was 1.06 mm at 25 #g/ml of
276 TABLE 2 Average differential migration. Subject No.
Average migration difference (mm) con troi-test
Skin test response to PPD Intermediate
2nd strength
"6 1 12 13 2 5 8 16
0.151 0.547 0.547 0.870 0.922 0.943 0.943 0.984
-+ , + + ----
+
11
1.000
--
--
15
1.005
--
+
1.026
--
+
10
1.026
--
--
9
1.036
+
14
3
+ ND + --
1.089
--
+
7
1.182
--
--
4
1.229
ND
ND = not done. Standard error of the average differential migration = 0.080 (15 d.f.).
PPD and 0.752 m m at 6.25 pg/ml (the standard error o f the differential concentration mean was 0.047, 15 d.f., P < 0.001 for difference between average differential migrations).
Effect of duration of culture on the migration of indicator cells incubated with culture supernatant Statistically there was a strong effect of culture time on m e d i a t o r activity. The results are given in fig. 2. There was a small b u t definite increase in the migration diameter in the control wells and a decrease in the test wells, although the difference between contr,~! and test was very well established after one day. These data were used to compare expressing the d a t a as mean differential migration diameters with the more conventional use o f percent inhibition in the area of migration. As shown in fig. 2, there is n o difference between these two m e t h o d s o f expressing the data.
Reproducibility of the microassay for leukocyte migration The data were quite consistent. The mean square o f all interaction terms with days (from a subject (16) × concentration (2) X replicates (2) X days (6) analysis o f variance, which ignores some aspects o f the design) had t h e value o f 0.0615 m m 2 (315 d.f.). The largest term 0.0968 (75 d.f.) was the interaction of subjects with days, which would be expected to be s o m e w h a t larger because o f the non-responsiveness o f a few subjects. The value o f the 4-
277
CONTROL
TEST •
•
i"
6-
CONTROL-TEST
2O io I 1
I 2
I 3
I 4
I 5
I 6
I
7
DAYS OF CULTURE
Fig. 2. Effect of duration of culture on the migration of indicator cells incubated with culture supernatant. factor interaction, 0.0480 (75 d.f.), was consistent with what would be expected from quadruplicate tests, 0.037 (2304 d.f.). (The mean square of 768 se~s of quadruplicate wells was 0.0740. The variance of the difference b e t w e e n two averages of 4 wells each was estimated as 1/2 the pooled mean square, 0.0740/2 = 0.0370.) The effect of replicate cultures was estimated from the pooled interaction o f replicate cultures with subjects, concentration, and subject X concentration (31 d.f.). The effect was estimated as equivalent to adding a random effect with standard deviation of 0.2 rnm. No significant difference could be ascribed to using migrating cells from two different individuals on the basis of a non-orthogGnal analysis of variance c o m p u t e d using program BMD1OV (Dixon, 1973).
Experiment B Effect o f antigen and cell concentration on the migration: o f indicator cells incubated with culture supernatent The differences between average control and test migration diameter was c o m p u t e d for each reader-culture combination. The average difference for the factors of interest are p l o t t e J in fig. 3. The results indicate that even quite small numbers of cells are effective ~:jven adequate concentrations o f antigen. What is n o t shown b y the figure is :,hat the cultures were n o t consis-
278 ..=.
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1.0 4x
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.8
~
.6
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.4
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.5 x 106cells/ml
1.0-
106cells/ml
l x 106 cells/ml
1.2 -
~
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