Cell-Mediated Immunity and Specific Serum Factors in Human Cancer: The
Leukocyte Adherence Inhibition Test 1,2
Annette Maluish and W. J. Halliday
3,4
SPECIFIC CELL-MEDIATED IMMUNITY
(CMI) in cancer is detectable by many different in
vitro techniques, of which lymphocyte cytotoxicity
against tumor cells is the most notable. This phenomenon seems a functional mechanism of tumor
immunity in vivo and, as particularly shown by the
Hellstr6ms (1, 2), it has facilitated the discovery of
serum factors which modify CM!. In both animals
and man, a circulating blocking factor (BF), capable
of interfering specifically with the lymphocyte-tumor
interaction in vitro, has been found in tumor-bearing
individuals; after successful tumor removal, BF
usually disappears and is replaced in the serum by
"unblocking" substances which cancel blocking
activity in serum mixtures.
Blocking and unblocking of antitumor CMI are of
practical importance as possible approaches to the
immunodiagnosis of cancer. However, established
techniques in the field of CMI are slow and cumbersome and thus unsuited to routine use. Simple, rapid
methods are needed.
The leukocyte adherence inhibition (LAI) technique, previously detailed for experimental murine
tumors (3, 4), was recently shown, in a small series of
human cases, to be capable of detecting tumor-specific
CMI and blocking by serum (5). The normal tendency
of blood leukocytes to adhere to glass was diminished
by the products of specific lymphocyte-antigen
interaction, and the inhibition was reversed by the
appropriate serum. In this paper, we show that the
method has given consistent results in a larger series
and has been used also to detect unblocking activity
in the sera of cancer patients with regressing tumors
or after successful tumor removal.
MATERIALS AND METHODS
Patients had malignant melanoma (10 cases),
adenocarcinoma of the colon and rectum (8 cases),
and adenocarcinoma of the breast (1 case); in addition, several patients had benign conditions.
Collection oj leukocvtes.-Heparinized venous blood
was allowed to stand in glass bottles and the leukocytes were separated as described in (5). Buffy coat
cells, obtained by centrifugation of blood, were also
used. For still obscure reasons, settling of blood in
plastic tubes yielded cells which were not active
in the LAI test. The final leukocyte suspension
contained, after washing, 2 X 10 7 cells/ml Eagle'S
basal medium (EBM) with fetal calf serum (FCS)
(3--5).
Serum.-Serum was separated from clotted blood
and diluted 1: 1 with EBM plus FCS before use.
Tumor extracts (antzgens).-Aqueous extracts were
prepared by homogenizing tumor tissue in phosphatebuffered saline, pH 7.4, and centrifuging (5). One
extract (C3) of a colonic carcinoma was made with
perchloric acid (6), followed by dialysis against
distilled water; this material was generously provided
by Dr W.H. Isbister. The extracts were arbitrarily
diluted 1: 5 with EBM before use. Their total protein
contents (Lowry method) differed widely, and no
attempt was made to standardize these or to investigate other dilutions. Initial protein contents were
as follows (in mg/m!): melanoma extract MI, 78;
M2, 4.8; colonic carcinoma extract Cl, 3.6; C2, not
done; C3, 0.2; breast carcinoma extract Bl, 3.0; B2,
2.2. All extracts were prepared in advance, tested for
nonspecific reactivity as described below, and stored
at -20° C; they were not autochthonous with
respect to any patients studied for CMI and blocking.
LA! test.-The technique was described in (3-5).
Duplicate mixtures of leukocytes, tumor antigen,
serum, and EBM plus FCS in the required combinations were made in a total volume of 0.2 ml, incubated
for 30 minutes at 37° C, and then introduced into
hemacytometers. After a further incubation of about
60 minutes, the cells were counted in predetermined
areas. The cover slips were floated off, the slides
rinsed, and the remaining adherent cells counted
in the same areas. All counting was done without
knowledge of the contents of the mixtures. Finally,
mean percent adherent cells and SE were calculated
for each mixture, and the significance of comparisons
was determined by the t test.
Where a positive result was reported (leukocytes
active, serum blocking or unblocking), there was a
Received October 16, 1973; accepted January 28, 1974.
Supported by the National Health and Medical R<:sear~h
Council, the Queensland Cancer Fund, and the Umverslty
Cancer Research Fund.
3 Department of Microbiology, University of Queensland,
St. Lucia, Brisbane, Australia.
4 We thank Dr. W. H. Isbister, Prof. G. J. A. Clunie, Dr.
J. H. Little, Dr. M. A. H. Gardner, Dr. N. C. Davis, and the
Queensland Melanoma Project for assistance in obtaining
material.
I
2
JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 52, NO.5, MAY 1974
1415
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SUMMARY-Blood leukocytes from patients with cancer
(malignant melanoma; adenocarcinoma of colon, rectum,
and breast) reacted with aqueous extracts of tumors of the
corresponding type, with the result that adherence of the
leu kocytes to glass was diminished. Leukocytes from normal individuals did not react. The leukocyte adherence inhibition (LAI) test could be completed in a few hours. Sera
from tumor-bearing patients blocked the LAI reaction of
their own leukocytes or leukocytes from other patients
with the same type of tumor. Serum blocking activity was
lost soon after surgical removal of the tumor; the patient's
serum then became unblocking. The LAI technique gave
consistent results in a series of patients, analogous to those
reported with the lymphocyte cytotoxicity test, and was
easier and quicker.-J Natl Cancer Inst 52: 1415-1420,1974.
1416
MALUISH AND HALLIDAY
highly significant difference between test and appropriate control (P<O.OI and usually <0.001).
RESULTS
Testing Extracts for Nonspecific Reactivity
Specific Antitumor eMI and Blocking
An example of a composite experiment with the
LAI test is shown in table 2. Mixtures 1, 2, and 3
illustrate simple CMI: The normal adherence of
melanoma patient W.T.'s leukocytes was markedly
reduced by an extract from an allogeneic melanoma
(compared with breast carcinoma antigen as a
control). This indication of tumor specificity is
supported by mixtures 5, 6, and 7, in which the
inhibitory activity of the antigens was reversed when
leukocytes from patient O.K. with breast carcinoma
were used. Mixture 4 exhibited blocking by W.T.'s
own serum; mixture 8 showed blocking by O.K.'s
own serum. Analogous results with other patients
were reported previously (5).
TABLE
I.-Effect of antigen extracts on normal leukocytes
LeUkocytes
Normal
Normal
Normal
Normal
(W.O.) ____
(W.O.) ____
(W.O.) ____
(W.O.) ____
Antigen (tumor
extract)
- ___________________
Melanoma (M1) _______
Melanoma (M2) _______
Oolonic carcinoma
Percent
adherence
(mean±sE)
47.
43.
42.
31.
1±2.
8±3.
7±2.
8± 1.
2
1*
4*
1t
(01).
Normal (W.O.) ____ Oolonic carcinoma
20. 6±2. 3t
(02).
Normal (W.H.) __ _
69.
Normal (W.H.) ___ Br~~;tc~;~i;;~m~------ 65.
(B1)t.
Normal (W.H.) _ _ _ Breast carcinoma
67.
(B2).
Normal (W.O.) ____ - __________________ _ 57.
Normal (W.O.) ____ Oolonic carcinoma
52.
3±2. 8
3±2. 7*
2±3. 0*
9±2. 4
9±2. 6*
(03) §.
"Not Significantly ditferent (rom control without antigen (P>O.3).
tSignificantly inhibited (P<O.Ol); I.e .. nonspecifically reactive.
tA mixture o( extracts (rom several similar tumors.
§Perchloric acid extract; all other antigens extracted with phosphatebutfered saline ("aqueous" extracts).
All patients with positively diagnosed progressive
cancer whose leukocytes and serum were examined by
the LAI technique showed detectable specific CMI
and serum blocking activity. Three additional melanoma patients were especially interesting, since they
were seen at the time of apparent tumor regression or
after tumor removal.
Patient C.A. had a melanoma on the chest. It
seemed in a state of regression and this was confirmed
histologically after excision. Blood taken at surgery
yielded reactive leukocytes, but the serum was not
blocking; when mixed with a known blocking serum,
C.A.'s serum restored LAI-i.e., it was unblocking
(table 3, A). The effect was not a result of simple
dilution of serum, since blocking serum mixed with
normal serum retained its activity.
Patient M.G. was studied only after removal of a
melanoma described histologically as "regressing."
Her serum was unblocking (table 3, B) 7 weeks after
excision; 19 weeks after surgery, it had lost all detectable activity.
Patient A.C. was examined several times at intervals
after removal of a melanoma. As shown in table 4,
her leukocytes were reactive against an allogeneic
melanoma antigen at all stages, but serum activity
went through a series of changes. Two weeks after
surgery, her serum was blocking and this activity
was lost at 7 weeks. At 12 and 17 weeks, her serum was
still not blocking, but it then had the ability to
abrogate (unblock) the blocking effect of her 2week serum (or of blocking serum from patient J.N.).
Findings With 22 Patients
Table 5 summarizes our total experience with all
patients examined to date. The only omissions are
reactions with unrelated antigens; these were consistently negative.
The list begins with those patients examined for
CMI, blocking, and unblocking on several occasions.
Detailed data for A.C., C.A., and M.G. are given
above (tables 3, 4). Melanoma patient A.C. has
retained active leukocytes (tested with 2 different
antigens) over several months' observation; after
tumor removal, her blocking serum, obtained only
once, was nevertheless tested many times against
allogeneic leukocytes from different patients with
melanoma and always had the same properties. Sera
obtained from A.C. on other occasions, and from C.A.
and M.G., were not blocking but were at times
unblocking (tables 3, 4). J.N. had disseminated
melanoma with continually active leukocytes and
blocking serum. S.D. was examined 7 days after
radiation therapy and behaved as did other tumorbearing patients (leukocytes active; serum blocking);
4 days later, the melanoma was removed and he was
given a blood transfusion. Two days after surgery, his
leukocytes were extremely adherent (>80%) and
inactive in LAI-a rare anomalous reaction-but his
serum was still blocking on this and a later occasion.
Four other melanoma patients had active leukocytes
and, in 2 cases, their sera were examined and found
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Tumor extracts were tested for their ability to bring
about nonspecific LAI with normal cells. Leukocytes
from normal individuals were incubated with and
without the various extracts and the percent adherence
was determined. All the melanoma and breast
carcinoma extracts were nonreactive, i.e., they did
not significantly affect normal leukocyte adherence
(table I). Two aqueous extracts of colonic carcinoma
were inhibitory and were therefore discarded; a
perchloric acid extract was not inhibitory to normal
leukocyte adherence. After an extract was shown to
have no nonspecific reactivity, it was assumed that
inhibitory activity with a tumor patient's leukocytes
was specific for tumor-associated antigen; this assumption was upheld by study of further controls with cells
from patients with unrelated tumors or nonmalignant
diseases.
Unblocking in LAI Tests
1417
LAI TEST IN HUMAN CANCER
TABLE
Mixture
No.
Leukocytes
cytes active with 2 appropriate antigens, and her
own serum was blocking with both antigens.
Finally, a very small number of nonmalignant
conditions evinced no reactivity with antigens thought
to have some possible relationship.
DISCUSSION
When the LAI test was first devised (3), we anticipated that it would be a convenient method of detecting the CMI developed against tumor-associated
antigens. Each step was intentionally made as uncomplicated as possible so that the test would be
technically simple and rapid. It has been gratifying
to find that antitumor CMI in animals and man can
indeed be detected by LAI, and that, in addition,
serum factors modify the reaction. The phenomena
2.-LAI by tumor antigen and blocking by patient's serum*
Antigen
Serum
Percent
adherence
(mean±sE)
L ______ Melanoma (W.T.) ______________________ NormaL __________ _
2 _______ Melanoma (W. T.) ___ Melanoma (Ml) _____ Normal ___________ _
77. 2±1. 8
30. 5± 1. 6
NormaL __________ _
3 _______ Melanoma (W.T.) ___ Breast carcinoma
(Bl)
4 _______ Melanoma (W.T.) ___ Melanoma (Ml) _____ Melanoma (W.T.) __ _
74. 2±2. 7
NormaL __________ _
68. 8±2. 2
Breast carcinoma
Normal ___________ _
(Bl)
Melanoma (M3) _____ Normal ___________ _
34. 7±2. 3
Breast carcinoma
(Bl)
74. 4±2. 0
5 _______ Breast carcinoma
(O.K.)
6_ ______ Breast carcinoma
(O.K.)
7 __ _ _ _ _ _ Breast carcinoma
(O.K.)
II. ______ Breast carcinoma
(O.K.)
Breast carcinoma
(O.K.)
75. 6±2. 7
73. 9±2. 2
Result
Leukocytes active with
Ml antigen; P<O.OOI.
Leukocytes inactive with
Bl antigen.
Serum W.T. blocking;
P<O.OOI.
Leukocytes active with
Bl antigen; P<O.OOI.
Leukocytes inactive with
M3 antigen.
Serum O.K. blocking;
P<O.OOI.
'Patients W. T. and O. K. had progressing tumors with secondary deposits.
TABLE
Leukocytes *
C.A.
C.A.
C.A.
C.A.
C.A.
A.C.
A.C.
A.C.
A.C.
A.C.
J.N.
J.N.
J.N.
J.N.
J.N.
3.-Unblocking of eMI by serum from patients with regressing tumor or after tumor removal
Antigen
Ml
Ml
Ml
Ml
Ml
Ml
Ml
Ml
Ml
Ml
Ml
Ml
Serumt
Percent
adherence
(mean±sE)
Result
A. Serum from patient C.A. with regressing melanoma
N
60. 9±2. 2
Leukocytes active; P<O.OOI.
N
47. 9±3. 0
A.C. + N
63. 7±3. 0
Serum A.C. blocking; P<O.OOI.
C.A. + N
45. 4±2. 6
Serum C.A. not blocking.
A.C. + C.A.
46. 3±4. 0
Serum C.A. unblocking.
B. Sera from patient M.G. after removal of regressing melanoma
N
60. 2±2. 2
Leukocytes active; P<O.OOI.
N
32. 9±2. 0
Serum A.C. blocking; P<O.OOI.
A.C. + N
65. 0±2. 4
Serum M.G.7 not blocking.
M.G.7 + N
35. 0±2. 1
Serum M.G.7 unblocking.
A.C. + M.G.7
37. 9±2. 3
N
61. 9±2. 5
Leukocytes active; P<O.Ol.
N
50. 6±2. 8
Serum J.N. blocking; P<O.OOI.
J.N. + N
66. 9±2. 6
Serum M. G .19 not blocking.
M.G.19 + N
51. 6±3. 6
Serum M.G.19 not unblocking .
J.N. + M.G.19
68. 1±2. 9
• Leukocytes from patient C.A. at removal of regressing melanoma; from patient A.C. 2 weeks after removal of melanoma; from patientJ.N. with disseminated
melanoma.
tN, normal; A.C., from patient A.C. 2 weeks after removal of melanoma; C.A., from patient C.A. at time of removal of regressing melanoma; M.O. 7 and
M.O. 19, from patient M.O. at 7 and 19 wk, respectively, after removal of regressing melanoma; J.N., from patient J.N. with disseminated melanoma.
Downloaded from http://jnci.oxfordjournals.org/ at Pennsylvania State University on September 18, 2016
to be blocking. W.T. had lymph node involvement
and a secondary growth in the lung. K.B. had had his
tumor successfully removed 4 years previously; no
cellular or humoral activity (blocking or unblocking)
was detectable.
Among 6 patients with carcinoma of the colon,
J.G. was examined twice (cells active and serum
blocking) and J.T. once (with similar results). Only
serum samples, taken at operation, were available
for the other 4, and these all contained BF. Rectal
and colonic carcinomas seemed to cross-react with
the antigen used, since 2 sera from patients with
diagnosed carcinoma of the rectum blocked leukocytes from a case of carcinoma of the colon in their
reaction with antigen C3.
The single patient with carcinoma of the breast
(widely metastasized and rapidly fatal) had leuko-
1418
MALUISH AND HALLIDAY
observed (especially the kinetics and specificity of
eMI and blocking) are remarkably similar to those
found by the Hellstroms and others (2, 7-11) in
lymphocyte cytotoxicity tests with living tumor cells.
This suggests that the aqueous extracts of tumors
used in the LAI test contain, in the solubilized state,
antigens with properties similar to those of tumor
cell membranes. Similar extracts have been employed
in macrophage migration inhibition (MMI) reactions
(12, 13). The observation that LAI reactions are
tumor-type specific in human cancer means, in
addition, that the extracts lack those antigens common
TABLE 4.-Sequential studies of leukocytes and serum from a single patient after tumor removal
Leukocytes*
Ml
Ml
Ml
Ml
Ml
Ml
Ml
Ml
Ml
Ml
Ml
Ml
Ml
Ml
Percent
adherence
(mean±sE)
Serumt
N
N
A.C.2+N
N
N
A.C.2+N
A.C.7+N
A.C.2+ A.C.7
N
N
A.C.2+N
A.C.12+N
A.C.2+A.C.12
N
N
J.N.+N
A.C.17+N
J.N.+A.C.17
Result
60. 2±2. 2
32. 9±2. 0
65. 0±2. 4
65. 2±2. 8
35. 8±3. 0
62. 8±2. 6
39. 8±2. 6
58. 1±2. 9
74. 0±2. 5
42. 6±2. 1
74. 0±3. 6
44. 9±3. 3
42. 3±3. 0
75. 1±2. 3
55. 4±2. 2
78. 8±1. 8
54. 0±2. 5
52. 7± 1. 9
Leukocytes A.C.2 active.
Serum A.C.2 blocking.
Leukocytes A.C.7 active.
Serum A.C.2 blocking.
Serum A.C.7 not blocking.
Serum A.C.7 not unblocking.
Leukocytes A.C.12 active.
Serum A.C.2 blocking.
Serum A.C.12 not blocking.
Serum A.C.12 unblocking.
Leukocytes A.C.17 active.
Serum J.N. blocking.
Serum A.C.17 not blocking.
Serum A.C.17 unblocking.
'From patient A.C., 2, 7, 12, and 17 weeks after removal of melanoma.
tN, normal; A.C.2, A.C.7, A.C.12, A.C.17, as for leukocytes; J.N., from patient with disseminated melanoma.
TABLE 5.-Summary of leukocyte activities and serum blocking activities of all patients*
Patient
A.C. ________
C.A. ________
M.G .. ______
J.N. _________
S.D .. ________
I.J. _________
W. T. ________
D.Z. ________
F.G. ________
K.B. ________
J.G. _________
J.T. _________
J.D. _________
T.D. ________
T. C._ _ _ _ _ __ _
L.R. ________
L.B. ________
W.O. ________
O.K. ________
B.S. _________
C.C .. _______
M.S ... ______
Description
Melanoma ______________
Melanoma ______________
Melanoma ______________
Melanoma ______________
Melanoma ______________
Melanoma ______________
Melanoma ______________
Melanoma ______________
Melanoma ______________
Melanoma ______________
Ca.11 colon ______________
Ca. colon _______________
Ca. colon _______________
Ca. colon _______________
Ca. colon_ _ __ _ _ __ _ _ _ __ _ _
Ca. colon _______________
Ca. rectum _____________
Ca. rectum _____________
Ca. breast ______________
Rectal abscess ___________
Seborrheic keratosis ______
Pyogenic granuloma _____
Specific
antigen used
Ml; M2
Ml
Ml
Ml; M3
Ml
Ml
Ml
Ml
Ml
Ml
C3
C3
C3
C3
C3
C3
C3
C3
Bl; B2
C3
Ml
Ml
Leukocytes
active with
specific
antigen
5
2
4
1; N.A.l§
1
1
1
1
N.A.l
2
1
Serum activity
Blocking with Blocking with
patient's own
allogeneic
Not blockingt
leukocytes
leukocytes t
1
1
3
1
3
4
1
1
2
1
1
1
2
1
1
1
1
1
1
1
1
N.A.l
N.A.l
'Patients A.C., C.A., M.G., and K.B. had clinically diagnosed melanomas successfully removed; all other cancer patients had progressing tumors of the
indicated type at time of testing. For further data 8ee text. Where a positive result Is reported (cols 4-jj), activity In LAl was highly significant (P<O.OI or,
usually, <0.001). Figures correspond to number of different samples tested; some sera were tested several times with different leukocytes and always gave the
same result.
tFrom patients with a similar type of tumor.
Uncludes unblocking; 8ee tables 3, 4.
§N.A.: not active.
IICa.: carcinoma.
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A.C.2
A.C.2
A.C.2
A.C.7
A.C.7
A.C.7
A.C.7
A.C.7
A.C.12
A.C.12
A.C.12
A.C.12
A.C.12
A.C.17
A.C.17
A.C.17
A.C.17
A.C.17
Antigen
LAI TEST IN HUMAN CANCER
Antigenic cross-reactivity in vitro between tumors
of the same histologic type or origin has been reported,
not only in lymphocyte cytotoxicity tests (10, 11) but
also with serum cytotoxicity (18) and immunofluorescent tests for antibody (18, 19). This is an
important property of practical value in immunodiagnosis. Those tests relying on individually tumorspecific antigens (20), or on a common antigen found
in many or all types of cancers (14, 21), are restricted
in their application.
The sequence of changes in serum reactivity seen
with patient AC. is thought to depend on removal of
her tumor as a source of antigen. If BF is an antigenantibody complex (22, 23), serum blocking would
soon disappear as antigen is catabolized, to be replaced by free antibody with unblocking properties.
The latter may prevent BF from acting in vitro by
binding to its antigenic sites. In the period of serum
inactivity, between the blocking and unblocking
phases, BF is presumably exactly neutralized by antibody in vivo and neither is detectable. These observations do not necessarily correspond to those made by
techniques which detect various other antitumor
antibodies (18-20), but they seem to have the
advantage of being logically associated with growth
and removal of tumors.
The kinetics of development and disappearance of
specific BF have been noted by other investigators
using the lymphocyte cytotoxicity technique (7, 8, 10,
24). The constant association ofBF with tumor growth
suggests that it functions in vivo by preventing lymphocytes from attacking tumor cells and that mani pulation ofBF activity might be useful in immunotherapy
of cancer. The loss of BF after tumor removal or
natural regression led initially to the notion that BF
contains tumor antigen (22). In the studies reported
here (table 4), serum from a patient with melanoma
remained detectably blocking for at least 2 weeks
after surgery. Blocking activity was not apparent
after 7 weeks, whereas CMI persisted for a much
longer time, accompanied by unblocking activity in
the serum. All activity was absent from another
patient 4 years after tumor excision (K.B., table 5). In
immediate practical applications, a rapid and reliable
technique for detecting specific antitumor CMI and
BF should have great value in diagnosis and monitoring the results of therapy. The persistence of BF
during tumor growth and its loss after successful
surgery would be especially useful in prognosis.
In a series of patients with known cancer, positive·
results were obtained in all tests satisfactorily completed (table 5). The anomalous finding with S.D.'s
cells may be related to postoperative depression of
eMI (25). The LAI test has thus revealed cellular
and humoral immune reactions which corresponded
to the patient'S tumor status in terms of presence or
absence of tumor of a certain type. Although changes
related to degree of involvement, metastasis, or
tumor size have not yet been investigated systematically, patients with small and large tumors and
extensive metastases or none have all given clearly
positive results. An aspect of this work still under investigation concerns the quantitation of CMI and
Downloaded from http://jnci.oxfordjournals.org/ at Pennsylvania State University on September 18, 2016
to a variety of tumors, e.g., the basic protein antigen
of Caspary and Field (14). The behavior of tumor
extracts suggests also that they contain no significant
quantities of BF, though these substances are present
in tumor tissues and easily eluted therefrom (15).
A possible interfering factor, which might be anticipated to emerge from the use of allogeneic extracts
and leukocytes, is reactivity against alloantigens such
as histocompatibility (HL-A) antigens. It might be
argued that leukocytes from patient A could react
with tumor extract from patient B because of previous
exposure to B-type alloantigens through blood transfusion or pregnancy. Our findings so far seem tumorspecific, both for CMI and especially in the regular
observation of blocking by patients' sera. One would
not expect blocking in simple allosensitization, though
the situation in pregnancy might be more complex.
Some technical advantages of the LAI technique
were summarized in (3). In the application to human
cancer patients; it is convenient to use stored tumor
extracts as antigens and not to rely on cultivated
tumor cells or the patient's Own tumor tissue. CMI
and BF can be determined by the examination of
blood specimens only; if a stock of appropriate
antigen and a source of fresh reactive leukocytes are
both available, BF determinations are readily performed with only the patients' sera.
A further unexpected advantage of the LAI technique is that the short incubation times may allow
the avoidance of some nonspecific effects found with
other methods. For example, MMI reactions are
susceptible to changes in serum and antigen concentrations (16), which affect the normal migration of
cells over many hours. Although the methods chosen
f,?r preparing tumor extracts were quite arbitrary and
YIelded products variable in protein concentration and
(we assume) in specific tumor antigen concentration,
all the extracts prepared have been suitable for use in
the test, except for aqueous extracts of colonic
carcinomas. These were nonspecifically reactive,
probably because of contamination by enteric bacteria
or their endotoxins. The immediate solution to this
problem-use of an extract prepared with perchloric
acid-is not regarded as entirely desirable and we are
attempting to produce better aqueous extracts;
expenence with the other tumors suggests that
aqueous e~tracts would be specific for tumor type,
whereas thiS may not be true for extracts prepared in
ot~er ways. For example, the colonic carcinoembryomc antIgen (CEA) appears to be associated with a
variety of tumors, from which it is prepared by
perchloric acid extraction (6). Our extract C3
p~obably contains this antigen and others; C3 reacted
WIth leukocytes from patients with colonic and rectal
carcinomas (table 5) but not with cells from several
patients with melanoma or breast carcinoma. We do
not know whether CEA stimulates specific CMI
detectable in vitro by the LAI test.
Only one dilution of serum has been used routinely
~o test for blocking, which was frequently complete;
It seems unlikely that serum dilution is a critical factor
in ~~I tests, as it is in blocking of lymphocyte cytotOXICIty (17).
1419
1420
MALUISH AND HALLIDAY
REFERENCES
(1) HELLSTROM I, HELLSTROM KE, EVANS CA, et al: Serummediated protection of neoplastic cells from inhibition
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Downloaded from http://jnci.oxfordjournals.org/ at Pennsylvania State University on September 18, 2016
BF. With a slightly modified procedure (26), eMI
can be detected with small (0.5 ml) samples of blood,
by a 2-stage technique which allows the separation of
a soluble factor from the cell-antigen mixture. This
factor is then quantitated by its effect on the adherence of normal cells (human blood leukocytes or
mouse peritoneal exudate cells). Supernatants may
be stored frozen and tested simultaneously with the
same batch of normal cells. In this way, the variability
in the adhering capabilities of cells at different times
or from different individuals is avoided and one can
measure changes in primary lymphocyte-antigen
reactivity, resulting either from variations in sensitized lymphocytes in cell populations used or from
BF in sera added to the mixtures.
The relationship of LAI to other in vitro manifestations of eMI is unclear. A soluble factor is
formed in <30 minutes by sensitized leukocytes exposed to appropriate tumor antigens (26). This material may be similar to migration inhibition factor
(MIF) , but LAI appears quite different from the
increased adherence of macrophages, incubated with
MIF-rich culture supernatants for several days (27).
The LAI technique seems applicable to the study of
eMI, BF, and other serum factors in human cancer
and, by virtue of its reliability and rapidity, it should
become a useful diagnostic and prognostic aid.