CELLULAR

An

23, 334-341 (1976)

IMMUNOLOGY

Evaluation of the Test as a Correlate

Peripheral Leukocyte Migration inhibition of Delayed Cutaneous Hypersensitivity 1

E. W. RAMSEY, L .J. BRANDES, K. H. A. Departments

AND

G.

J.

GOLDENBERG

of Surgery

and Medicine, The University of Manitoba The Manitoba Institute of Ccl1 Biology, Winnipeg, Manitoba, R3E OV9, Calzada Received January

The leukocyte

JACOB,

migration

inhibition

19, 1976

test was used to study the in vitro

PPD of peripheral blood leukocytes from six Mantoux-positive negative volunteers. All tests were performed the test. Results with the two techniques

response to

and five Mantoux-

using both micro and macro versions of were usually, but not always similar.

Subjects were tested weekly for 6 weeks and variation was noted from week to week. Leukocytes

and

from

in the degr’ee of inhibition

Mantoux-positive

subjects som,etimes

did not have significant migration inhibition while those from Mantoux-negative subjects sometimes did. Despite this variation the Mantoux-positive subjects both as a group and in most cases individually, had significantly greater mean migration inhibition than the Mantoux-negative subjects. These results suggest that sequential may provide a more meaningful index of testing rather than a single determination cellular

immunity.

INTRODUCTION The inhibition of peripheral blood leukocyte migration by various antigens has been widely used as an in vitro assay of delayed hypersensitivity (l-9). Although a number of reports have claimed it to be a reliable and specific indicator of cellular immunity (l-5)) others have reported a lack of confidence in the reproducibility and interpretation of the test (6-9). Several techniques have been used to assess migration inhibition (1, 4, 10, 1 l), but the migration of leukocytes from capillary tubes has been that most widely reported. The use of microcapillaries (4) instead of the more commonly used hematocrit capillaries has the considerable advantage of allowing a large number of replicates from small volumes of blood. It has also been suggested that this microtest is more sensitive (12). A feature not stressed in previous reports has been the variation in reactivity when subjects were tested on more than one occasion. Therefore the present study was undertaken to compare micro and macro techniques and to determine if week to week variation occurred. Tests were performed in Mantoux-positive and negative subjects usin, u tuberculin purified protein derviative (PPD) as the test antigen. MATERIALS

AND

METHODS

Antigen Preservative-free PPD, 2000 pg/ml protein concentration tories) was used as the test antigen for all in vitro tests. 1 This

work

was supported

by a grant

from 334

Copyright All rights

0 1976 by Academic Press. Inc. of reproduction in any farm reserved.

the National

Cancer

(Connaught Institute

Labora-

of Canada.

LMT

AS

A

CORRELATE

OF DELAYED

IIYPERSENSITIVITY

335

Sub jccts Six Mantoux-positive and five Mantoux-negative volunteers tive subjects were those with palpable induration 3 10 mm, dermal injection of 0.1 pg PPD (5 TU). Negative subjects tion to 5 pg PPD (250 TU) both before and after completion

were studied. Posi4s hr after intrashowed no induraof the study.

Leukocyte Preparation Heparinized peripheral venous blood was sedimented using Plasmagel (Laboratoire Roger Bellon, Neuilly, France), the leukocyte-rich supernatant was centrifuged at 1000 rpm for 10 min in a Sorvall GLC-I centrifuge and the leukocyte pellet was washed consecutively with minimal essential medium (Grand Island Biological Co., Grand Island, N.Y.), a red cell-lyzing solution consisting of 0.14 M ammonium chloride -0.017 hf Tris buffer (Sigma Chemical Co., St. Louis, MO.) at pH 7.2, and minimal essential medium, centrifuging after each step at 1000 rpm for 10 min. The final leukocyte pellet was resuspended in approximately nine times its volume of Medium 199 (Grand Island Biological Co.) containing 10% horse serum. Leukocyte Migration

Test (LMT)

The leukocyte suspension was divided in two parts, one being used for the microtest and the other for the macrotest. These tests were performed at the same time by independent operators. In the microtest, microcapillaries, length 6.4 cm, internal diameter 0.6 mm (Drummond Scientific Supplies) were used and in the macrotest, hematocrit capillaries, length 7.5 cm, internal diameter 1.2 mm, (Capilets, American Hospital Supply Corporation). Microcapillaries were filled by capillary action and one end plugged with paraffin wax. Hematocit capillaries previously heat sealed at one end were filled with a syringe connected to a 20 gauge needle. The capillaries were centrifuged at 1000 rpm for 10 min in a Sorvall GLC-I centrifuge, cut below the cell fluid interface and mounted into Mackaness chambers, the microcapillaries in pairs and the macrocapillaries singly. Control chambers were filled with Medium 199 containing 10% horse serum, 50 units/ml penicillin and 50 pg/ml streptomycin ; test chambers with the identical medium contained PPD in concentrations of 5, 15, or 50 pg/ml. After sealing the chambers with cover slips they were incubated at 37°C for 20 hr and the areas of migration were measured by projection microscopy and planimetry. In every experiment four capillaries were set up in control medium and at each concentration of PPD. Tracing and planimetry were carried out without knowledge of the subjects skin test status. Frequency of Testing The 11 subjects were tested weekly for 6 weeks at concentrations of 5 and 50 rg/ml PPD. In addition three of the Mantoux-positive and three of the Mantoux-negative subjects were tested weekly for 6 weeks at 15 pg/ml. The in vitro testing was started within 2 weeks after completion of skin testing.

336

RAMSEY

ET AI..

Expression of Results Results were analyzed in two ways to see which correlated better with test status. Percentage inhibition was calculated using the formula:

skin

area of migration in presence of PPD X 100 area of migration in control chamber c >’ Inhibition of 20% or greater was considered a positive result. Results were also analyzed statistically by comparing the area of migration in the chambers containing PPD with those of the controls using a two-tailed Student’s t-test. A p value of < 0.05 was considered a positive result. Y. Inhibition

= 100 -

RESULTS Comparison of Micro and Macro Techniques Although results with both techniques were often similar, some discrepancies in concurrent micro- and macrotests did occur. This is illustrated by Table 1 which shows the results of weekly tests in one skin test-positive individual using both tests at 50 g/ml PPD. While results were similar on weeks 3, 5, and 6, considerable differences were present on the other weeks. A total of 168 tests were performed using both techniques. In 29 of these, greater than 20% inhibition was found with the macro test but not with the micro, while in 12, migration inhibition greater than 20% was found with the micro test but not with the macro test. Week to Week Variation Variation in the degree of inhibition was often seen from week to week in both groups. Table 2 shows percentage inhibition each week of all subjects tested with the micro technique at 50 pg/ml PPD. Testing Mantoux-positive subject 1 for example on week 4 gave 41.8% inhibition and two weeks later 1% stimulation of leukocyte migration. A single test such as the result obtained on week 6 would therefore have indicated lack of immunity to PPD, although this subject had a mean migration inhibition of 23.2 * 6.17’0 over the 6 week period. Similarly, a TABLE Percentage

1

Migration Inhibition with Micro and Macro Subject 1 at 50 rg/ml PPD y0 Migration

Week Micro 1 2 3 4 5 6 a Mean f

SE of quadruplicate

16.1 21.9 25.5 41.8 35.1 - 1.9 determinations.

f f f f f f

Tests in

inhibition Macro

0.4a 5.9 2.8 2.2 1.7 4.7

35.6 36.2 25.0 17.4 36.8 1.0

f 2.3 f 2.6 f 2.7 f 4.2 f 3.1 f 3.5

Mean f

7 8 9 10 11

SE

Mantoux-negative

SE

11.4 f

6.1 2.3 0.0 20.9 27.5

17.9 f

5.4

11.1 f

14.8 15.2 8.6 11.3 5.8

20.0 f

1.8

11.4 f

8.5 4.5 18.8 19.7 5.6 3.3

20.7 z!z 3.0

-

0.9 f

.-.

4.2

5.8

-1.1 -1.4 -4.8 23.0 -11.1

22.6 f

-

11.2 xk 6.0

21.0 1.8 -7.6 21.3 19.7

23.1 rt 3.0

26.2 13.1

15.1 23.3

28.8 14.9

18.2 8.4

21.1 13.3

56

Mean f

21.2 18.4 24.7

18.6 22.7 13.9

27.1 16.9 11.2

24.6 26.2 21.1

16.4 20.0 20.4

2 3 4

Week 5

PPD

35.1

Week 4

Test at 50 pg/mI

41.8

Week 3

Micro

2

25.5

2.6

Week 2

with

TABLE Inhibition

21.9

1.3

Week 1

Migration

16.1

Mantoux-positive 1

Skin test status

Percentage

4.6 f

-0.6 6.0 -1.0 5.4 13.0

14.4 f

-1.0 4.0 21.4 22.7 21.4 17.6

2.7

4.2

Week 6

f f f f f f

f f f f f 8.4 f

8.1 4.7 2.3 16.7 10.1

19.8 f

23.2 18.7 20.9 18.9 21.8 15.1

2.5

3.5 2.3 4.0 3.0 5.5

1.2

6.1 3.3 1.3 2.2 2.1 2.0

Mean f SE (all tests) ,?

g

B 2

s

5

8 E F

% *

338

RAMSEY

ET

TABLE

Skin test status

Mean Percentage

Migration

“/e Migration inhibition Antigen concentration

microtest (rg/ml)

5

15

AL.

3 Inhibition

of 6 Weekly

Tests

c/0 Migration inhibition Antigen concentration

50

5

macrotest (rig/ml)

15

50

Mantoux-positive 1 2 3 4 5 6 Group mean &SE

10.3 7.6 11.9 9.5 13.9 4.2

f f f f f zt

9.6 f

3.7 3.2 1.4 2.9 2.4 1.3

II.8

f

3.1

23.2 18.7 20.9 18.9 21.8 15.1

16.1 f 12.5 f

5.6 4.1

1.1

13.5 & 2.4

19.5 f

IO.2 f 5.8 f 5.3 f

8.1 4.7 2.3 16.7 10.1

6.1 3.3 1.3 2.2 2.1 & 2.0 dz zk r!z zk rt

14.3 8.1 12.3 13.1 17.0 8.4

f f r!z zt f f

2.7 1.9 3.3 3.3 4.0 3.9

1.4

12.2 zt 1.3

rt 3.5 f 2.3 f 4.0 zk 3.0 zt 5.5

0.4 f 2.2 5.6 f 3.2 7.8 iz 3.6

8.4 zk 1.8

4.2 rt 1.7

16.4 zt 3.7

15.7 * 5.4 10.1 f 3.3

25.3 25.8 23.8 24.2 30.0 21.3

f f f f f f

5.8 3.2 4.0 3.6 5.6 3.6

14.0 f

24.8 f

1.7

2.4

Mantoux-negative 7 8 9 10 11 Group mean *SE Pa

I.7 -5.8 -3.8 -0.9 2.1

ff * f f

1.8 2.0 2.5 2.9 1.6

-1.3

f

1.1

An evaluation of the peripheral leukocyte migration inhibition test as a correlate of delayed cutaneous hypersensitivity.

CELLULAR An 23, 334-341 (1976) IMMUNOLOGY Evaluation of the Test as a Correlate Peripheral Leukocyte Migration inhibition of Delayed Cutaneous Hy...
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