32
with 1% glutamine, 15% fetal calf serum, penicillin
(100 units/ml), and streptomycin (0.1 mg/m}).
Five-m] 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 hr the cells were harvested,
and the numberof transformed lymphocytes
(blastlike cells) was determined as follows. The
cells were prepared for counting by the method
of Stewart and Ingram.*! A 1-ml aliquot of each
culture was treated with a proteolytic enzyme
(pronase) to remove cellular 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?#2 had shown that the transformed
cells had nuclei larger than 47 cubic microns. The
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nique, the leukocytes removed from the buffy coat
are predominantly lymphocytes, but with varying
fractions of other leukocytes, principally neutrophils. Although the total numberof 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 hr, when the final counts were done,practically
all neutrophils had disappeared from the cultures
so that the percentage transformation of lympho-
cytes was notsignificantly affected by this variable.
Serum Proteins.
Serum was collected from
non-heparinized aliquots of blood from each in-
dividual. Total serum proteins (g/100 ml) were
determined with a refractometer (American
Optical-TS). Separation of serum proteins into
albumenandalpha-1, 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, tonic strength 0.075) was used with
a run separation of 17 min at 100 volts. The protein bands werestained with Ponceau-S dye and
then quantified by using a Beckman/Spinco Analytrol with a microzone scanning attachment.
Serum Immunoglobulins.
Immunodiffusion
procedures for the determination of immunoglobulins IgA, IgD, IgG, IgM, and kappa and
lambdalight chains were carried out by Dr. John
L. Fahey and Dr. Roy Woods of the National
Cancer Institute Immunoglobulin Center (Springfield, Virginia). The technique used for quantify-
4
Y= 72.47 ~0.24x
20 —
=
t
10
percent transformation was obtained by compar-
ing the numberoflarger cells with the total number of cells present. With the above culture tech-
DO UNEXPOSED
¥=70.20-0.16x
@---e EXPOSED
20
{
30
_t
40
AGE
50
i
|
60
70
80
Figure 34. Age-related change in lymphocyte transformation in peripheral blood cultures showing the mean percent transformation for each decade with standard
deviation.
ing the serum immunoblobulins in antibody-agar
plates has been previously described.*3
Peripheral Blood Elements.
The enumeration
of peripheral blood elements was part of the
routine medical examination of the Marshallese
(see below, under Hematological Fndings). Leuko-
cyte counts?! were carried out electronically
(Coulter A counter). Platelet counts#> were done
by phase microscopy. Differential counts of leukocytes (200 cells) were performed on Wrightstained
smears. Hematocrits were determined by the microcapillary method*é and sedimentation rates by
the method of Wintrobe.*7
Statistical Analysis of Data.
An analysis of
variance was used to determine differences among
groups for age, sex, and radiation exposure. These
data were programmed and analyzed on a high
speed digital computor. Since sex differences were
not apparent, the results for males and females
were combined and each,criterion was analyzed
for age correlation (r value). Thelevel of significance (pf) of differences between the exposed and
unexposed groups (radiation effects) was determined; p values <0.05 are referred to as “‘signifi-
cant” in interpreting these findings.
Results
The results are summarized in Table 16, and
the values of the various criteria are plotted as a