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.