with 1% glutamine, 15% fetal calf serum, penicillin (100 units/ml), and streptomycin (0.1 mg/ml). Five-ml cultures were seeded with 106 leukocytes/ml, PHA M Difco (0.32 mg/ml culture) rs 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 Cagcer Institute Immunoglobulin Center (Springfield Qirginia). The technique used for quantify- 9000284 o—o Or UNEXPOSED ¥*70.20-0,16X% @---» EXPOSED ¥: 72.47-0.24x ios Figure 34. Age-related change in lymphocyte transforma- tion in peripheral blood cultures showing the mean per- ing the serum immunoblobulins in antibody-agar plates has been previously described.*9 Peripheral Blood Elements. The enumeration rr cent transformation for each decade with standard deviation. Ca. waa of peripheral blood elements was part of the routine medical examination of the Marshallese (see below, under Hematological Fndings). Leukocyte counts*4 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‘ and sedimentationrates by ' the method of Wintrobe.47 Statistical Analysis of Data. An analysis of variance was used to determinedifferences among § groupsfor age, sex, and radiation exposure. These — data were programmed andanalyzed 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 oye capes:peer 37°C. At exactly 72 hr the cells were harvested, and the number of transformed lymphocytes (blastlike cells) was determined asfollows. The cells were prepared for counting by the method of Stewart and Ingram.*! A 1-mlaliquot of each culture was treated with a proteolytic enzyme (pronase) to remove cellular debris and a cytoplasmicstripping agent(cetrimid)to release intact nuclei. The nuclei were counted and sized with a Coulter electronic counter (Model A). Previous experiments?? had shownthat the transformed cells had nuclei larger than 47 cubic microns. The percent transformation was obtained by comparing the numberoflarger cellswith 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 numberof cells was constant at the beginningof culture for each individual, the number of lymphocytes varied because of slight differences in differential counts. However, by 72 hr, when thefinal counts were done, practically all neutrophils had disappeared from the cultures so that the percentage transformation of lymphocytes was notsignificantly affected by this variable. Serum Proteins. Serum was collected from non-heparinized aliquots of blood from eachindividual. Total serum proteins (g/100 ml) were determined with a refractometer (American Optical-TS). Separation of serum proteins into albumen and alpha-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, ionic 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. cra arta was added, and the cultures were incubated at PERCENT TRANSFORMATION = ee ae —— 32 for age correlation (r value). Thelevel of signifi- cance(pf) of differences between the exposed and unexposed groups(radiation effects) was determined; f 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 4