rat,
IMMUNOHEMATOLOGICAL ASPECTS OF FALLOUT RADIATION
MATERIALS AND METHODS
In Table 1 the numbers of Ss on whom the
various tests were done are listed according to
age decades.
Lymphocyte cultures—Blood cultures were
set up as follows. The buffy coat was separated
from 5 ml. of heparinized blood by sedimentation and centrifugation. The culture medium
consisted of Eagle’s minimum essential medium
supplemented with 1% glutamine, 15% fetal
calf serum, penicillin
rise, PhD,
el Makar’
rapid move-
and levels of
sium (#°K).
ying degrees
led a means
‘ore for each
tests showed
lature aging
Je associated
we have ex-
examination
tatus in the
ese populaiging and/or
idies include
‘plication of
shytohemagture, quanti-
tins by elec-
ber of transformed lymphocytes (blast-like
cells) was determined as follows. The cells
were prepared for counting by the method of
Stewart and Ingram (1968). A I-ml. aliquot
of each culture was treated with a proteolytic
enzyme (pronase) to removecellular debris and
a cytoplasmic stripping agent (cetrimid) to release intact nuclei. The nuclei were counted
and sized with a Coulter electronic counter
(Model A). Previous experiments (Conard,
1969) had shown that the transformed cells
had nuclei which were larger than 47 cubic
microns. The percentage transformation was
obtained by comparing the number of larger
cells with the total number of cells present.
With the above culture technique, the leukocytes removed from the buffy coat are predominantly lymphocytes, but with varying fractions of other leukocytes, principally neutrophils. Although the total number of cells was
constant at the beginning of culture for each
individual, the number of lymphocytes varied
because of- slight differences in differential
counts. However, by 72 hours, when the final
Table 1. Numbers of Marshallese Ss Tested
in Various Studies,
mtrast to re-
1en be com-
to evaluate
and
hours the cells were harvested, and the num-
lies for imenumeration
the present
sed popula| group that
ects. Theree unexposed
sly to deterria with agdation. The
(100 units/ml),
streptomycin (0.1 mg/ml). Five-ml. cultures
were seeded with 10° leukocytes/ml, PHA M
Difco (0.32 mg/ml culture) was added and the
cultures were incubated at 37 C. At exactly 72
Age
Group
Lymphocyte
Transformation
UnexExposed
posed
Serum
Proteins
UnexExposed
posed
TmmunoGlobulins
UnexExposed
posed
Blood
Elements»
Unex- Exposed posed
13-20
21-30
31-40
41-50
51-60
61-70
71-80
11
11
25
19
15
12
9
iu
9
10
4
6
3
1
12
ll
26
19
.
12
8
ll
10
10
4
6
3
I
6
9
9
11
ii
12
7
4
2
2
2
3
2
1
29
16
20
11
5
ll
6
15
7
7
8
5
3
1
Total
102
44
105
45
65
16
98
46
sSexes were combined since the results on males and females showed no
significant differences.
bThe blood element studies were carried out in 1967, the others in 1968.
S012} 5h
29
counts were done, practically all neutrophils
had disappeared from the cultures so that the
percentage transformation of lymphocytes was
not significantly affected by this variable.
Serum proteins.—Serum wascollected from
non-heparinized aliquots of blood from each
individual. Total serum proteins (g/100 ml)
were determined with a refractometer (American Optical-TS). Separation of serum proteins
into albumen and alpha-1l, alpha-2, beta, and
gamma globulin fractions was done by microelectrophoresis with strips of cellulose acetate
(Phoroslides) and a Millipore cell (Millipore
Corp.). Barbitol buffer (pH 8.6, ionic strength
0.075) was used with a run separation of 17
min. at 100 volts. The protein bands were
stained with Ponceau-S dye and then quantified
by using a Beckman/Spinco Analytrol with a
micozone scanning attachment.
Serum immunoglobulins—Immunodiffusion
procedures for the determination of immunoglobulins IgA, IgD, IgG, IgM, and Kappa and
lambda light chains were carried out by Dr.
John L. Fahey and Dr. Roy Woods of the National Cancer Institute Immunoglobulin Center
(Springfield, Va.). The technique used for
quantifying the serum immunoglobulins in
antibody-agar plates has been previously described (Fahey & McKelvey, 1965).
Peripheral blood elements—-The enumeration of peripheral blood elements was part of
the routine medical examinations of the Marshallese. Leukocyte counts (Richar & Breakell,
1959) were carried out electronically (Coulter
A counter). Platelet counts (Bull, Schneiderman, & Brecher, 1965) were done by phase
microscopy. Differential counts of leukocytes
(200 cells) were performed on Wright stained
smears. Hematocrits were determined by the
micro-capillary method (Strumia, Sample &
Hart, 1954), and sedimentation rates by the
method of Wintrobe (1967).
Statistical analysis of data—An analysis of
variance was used to determine differences
among groups for age, sex, and radiation ex-
posure. These data
analyzed on a high
Since sex differences
sexes were combined
were programmed and
speed digital computor.
were not apparent, the
and each criterion anal-
yzed for age correlation (r value). The level of
significance (p) of differences in the group
exposed to fallout compared with the unexposed
group (radiation effects) was expressed.