Daily variation in circulating lymphocyte counts, T and B proportions and responsiveness to phytohemagglutinin

Life Sciences, Vol. 21, pp . Printed In The U.S .A.

793-802

Perganon Press

DAILY VARIATION IN CIRCULATING LYMPHOCYTE COUNTS, T AND B PROPORTIONS AND RESPONSIVENESS TO PHYTOHEMAGGLUTININ P.T . Fan, D .T .Y . Yu, P.J . Clemente, G . Opelz, L . Goldberg and R. Bluestone With the technical assistance of Sharon Brady From the Medical and Research Services, VA Wadsworth Hospital Center, Wilshire and Sawtelle Blvds ., Los Angeles, California 90073 ; and the Department of Medicine, UCLA School of Medicine, Los Angeles, California 90024 . (Received in final form August 11, 1977) Summary Peripheral blood was obtained from six healthy individuals over five consecutive days under uniform conditions and the total lymphocyte counts, T and B proportions, and response to phythohemagglutinin (PHA) were determined . The daily variation in T lymphocytes as measured by the spontaneous sheep erythrocyte (SRBC) assay was much greater when total T concentrations rather than T percentages were There was considerable daily variation in PHA responsivecompared . ness and in the percentages of cells bearing Fc and C3 receptors and Cryopreservation did not affect the surface immunoglobulin (SIg) . proportions of T and B lymphocytes although it resulted in a significant enhancement of PHA responsiveness following the freeze-thaw procedure . The significance of these results is discussed . The enumeration of circulating lymphocytes, their ratios of T and B cells and their responses to phytohemagglutinin (PHA) stimulation are widely applied in clinical immunological studies (1,2) . These assays are frequently carried The values out either on fresh samples or on cells stored by cryopreservation . obtained from patients are commonly compered to those of healthy individuals. l. The reHowever, the "standardization" of normal values remains a problem: sults of T and B determinations in the peripheral blood can be expressed either as percentages of total lymphocytes or as absolute concentrations per blood volume . It has been repeatedly emphasized that when one group of subjects is compared to another, the absolute concentration should be used rather than the However, day to day variations within single individuals percentage (2-5) . have not been quantitated, and it is therefore not clear whether the percentages or the absolute concentrations will more accurately reflect changes in the 2. It is well accepted that the PHA immune status during multiple sampling . response of lymphocytes from the same subject varies considerably from day to Some of the physiological factors which affect mitogen reday .(see Ref . 23) . sponse have been recently elucidated . The variation may be much less if these 3. Cryopreservation permit stimultaneous testing of factors can be minimized . The effect cell samples which have been collected over a period of time (6,7) . of day to day variations within an individual on the validity of such comparisons has not been studied. Materials and Methods Subjects and Design of Experiment The participants were six healthy male white volunteers from 23 to 30 793-

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years of age . They were nonsmokers and were taking no medications within a month of the time of study . During five consecutive days, between 8 and 9 a .m ., 40 ml of blood was collected in preservative-free heparin by venipuncture . The subjects did not undergo any exercise and rested for 10 to 15 minutes in a sitting position before venipuneture . Resting pulse rates were taken and they varied negligibly in the same individual within the period of study . Hematological Studies Leukocyte counts were done in triplicate in hemocytometers . Differential counts were obtained by counting 200 cells from a Wright-stained peripheral blood smear . Total lymphocyte counts were computed from the leukocyte counts and the differentials . Preparation of Cells Within 15 minutes after collection, the whole blood was diluted three times with NaCl-EDTA . The cell suspensions were carefully layered onto a Ficoll-Hypaque gradient (density 1 .077) and centrifuged at 600g for 40 min. The cells at the interface were aspirated and washed twice in Hanks' balanced salt solution (HESS) (Gioco, Grand Island, N .Y .) . Freezing and Thawing of Cell Suspensions Cells to be frozen were suspended at 2 x 10 6/ml concentration in McCoy's medium containing 10% dimethyl sulfoxide (Gioco, Grand Island, N .Y .) . The cell suspensions were dispersed into Beckman microcentrifuge tubes, placed in a -80° C freezer for 24 hours, and then transferred to a -196°C liquid nitrogen tank (6) . Four to 6 weeks later, they were rapidly thawed in a 50°C water bath, immediately resuspended and washed twice in medium 199 (Gioco, Grand Island, N .Y .) . 65 .0 + 5 .2% intact cells were recovered and these were 91 _+ 1 .1% viable by trypan blue dye exclusion . Spontaneous SRBC Rosette Assay The details of this method of preparing sheep red blood cell (SRBC) rosettes have been previously described (8) . Briefly, lymphocytes were suspended in HBSS to a final concentration of 5 x 106 per ml . SRBC were washed three times with saline and resuspended to a concentration of 80 x 106 per ml in Hanks' with 10% heat inactivated fetal calf serum (Gioco, Grand Island, N.Y .) which had been adsorbed twice with SRBC previously . 0 .1 ml of lymphocytes was mixed with 0 .1 ml of SRBC suspension (ratio of 16 SRBC : 1 lymphocyte) in 10 x 75 mm glass tubes and centrifuged at 200 g for 10 min . The tubes were kept at 4°C overnight and then resuspended by vertical rotation . Three hundred cells were counted using a hemocytometer. An SRBC rosette was defined as a lymphocyte surrounded by 3 or more SRBC . The result was expressed as the percentage of lymphocytes forming rosettes . Each sample was assayed in triplicate . Surface Immunoglobulin - (SIg) Staining Lymphocytes were incubated at 37 0 C for 30 min . and washed twice with prewarmed RPMI 1640 (Gioco, Grand Island, N .Y .) . 3 x 106 lymphocytes were incubated with 0 .025 ml of FITC-conjugated goat polyvalent anti-human immunoglob ulin (Meloy Lab, Springfield, Va .) at 4°C for 30 min . (9) . Cells were washed three times in cold phosphate-buffered saline with 0 .1% sodium azide. Immunofluorescent cells were identified by epifluorescent with phase contrast microscopy (Zeiss, Ultraphot) . A minimum of 200 cells were counted. Monocytes were identified by their ability to ingest latex particles in vitro as reported previously (10) .

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Aggregated Immunoglobulin - SIg Receptor Assay This procedure identified lymphocytes bearing the Fc-receptor as well as those bearing surface immunoglobulin . The details have been described previously (10) 3 x 106 lymphocytes were incubated with 0 .5 ml of heat-aggregated After washing human gammaglobulin (Pentax, Kankakee, Ill .) for 30 min. at 40 C . twice, 0 .025 ml of undiluted FITC conjugated polyvalent goat anti-human immunoFollowing a 30 min . incubaglobulin (Meloy Lab, Springfield, VA .) were added. tion at 40C, the cells were washed three times and the percent of fluorescent cells determined .

Complement (C) Receptor Assay The method is adapted from the rosette assay for detecting complement receptor sites using complement-coated zymosan beads reported by (11) and deZymosan particles (Sigma, St . Louis, scribed by us in detail elsewhere (21) . Mo .) were sensitized with complement by incubation with fresh human serum. 0 .02 ml of lymphocytes (5 x 106 per ml in Hanks') were mixed in 6 x 50 mm glass tubes . The mixture was incubated at 37 0C for 10 min ., centrifuged at 200g for 10 min., and kept at 40C for 30 min . The suspension was gently resuspended by Pasteur pipette, one drop placed into a hemocytometer, and 200 lymphocytes were counted . All tests were performed in triplicate and the results expressed as A C-rosette was defined as a percentages of lymphocytes forming rosettes . lymphocyte surrounded by two or more zymosan granules under these conditions . PHA Stimulation

The details of the method have been reported elsewhere (13) . Briefly, 5 x 10 4 lymphocytes in 0 .1 ml medium 199 with 20% male AB plasma were mixed with 0.01 ml of serial twofold dilutions of phytohemagglutinin-M (PHA-M ; Difco, Detroit, Mich .) and incubated at 370C for 72h. Tritiated thymidine (0 .8 Ci ; sp . act 22 ci/mMol ; Schwartz/Mann Div. Becton, Dickinson, Orangeburg, N.Y .) was added for an additional l6h and the cultures were harvested. The trichloroacetic acid-precipitable H3 -thymidine was quantified by counting in a Searle Scintillation Counter (Amersham/Searle Corp ., Arlington Hts ., Ill .) . All cultures were performed in triplicate and the maximal PHA stimulation expressed as mean t SEM counts per minute . Statistical Calculations

To allow direct comparison of the variability of different lymphocyte parameters, the values obtained on successive days were expressed as percentages of those of the first day . The means and standard deviations of these percentages were calculated for each subject . The standard deviation was used as the index of the day to day variation of the different lymphocyte parameters . Further To evaluate the efcomparisons were made using the Student's t distribution . fect of cryopreservation on lymphocyte parameters, results obtained before and after the freeze-thaw procedure were compared by paired t-test analysis . Correlation between various lymphocyte parameters was assessed by measuring the linear correlation and expressed as the correlation coefficient . Results Total Lymphocyte Counts and T and B Surface Markers The results of the total lymphocyte counts,.SRBC rosette, AggIg-SIg receptor, SIg, C-receptor percentage, and total SRBC rosette concentrations obtained from one subject (D .R .) over five consecutive days are shown in Table 1 . These are representative of the group, the means and standard deviations of which are The marked fall in total lymphocyte concentration on Day 2 in subdisplayed . ject D.R . was not seen in any other subject and could not be attributed to the

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effect of phlebotomy on the preceding day. The same data are also shown with the results of successive days expressed as a percentage of Day 1 . The most striking finding was that the standard deviation of the percentages of SRBC rosettes was much less than that of the absolute concentrations (5 .8 + 1.3 vs 31 .5 + 3 .8) . This was highly significant (p<0 .001) . There were no significant differences between S .D .'s of the AggIg-SIg receptor, SIg or C-receptor cells (p>0 .05) . S .D .'s of the AggIg-SIg receptor and SIg percentages were significantly greater than that of the SRBC rosette percentages (p <0 .01 ; p<0 .05) . There was no correlation between the variations in SRBC rosette percentages and that of the AggIg-SIg, SIg or C-receptor surface markers . PHA Stimulation The maximal response of the lymphocytes to PHA stimulation varied considerably from day to day in the six subjects (average adjusted S.D . 22 .9 + 5 .6 (Table 4)) . There was no correlation between this variation and that of the SRBC rosette percentages . The result of one subject is shown in Table 2 . (Correlation coefficient r = -0 .38) . The correlation coefficients for the six subjects were -0 .38, -0 .01, 0 .24, -0 .44, -0 .51 and 0 .27 . Neither were the variations of PHA response related to those of the percentages of Agg-Ig-SIg, SIg or C-receptor cells . TABLE 2 Daily Variation In Maximal PHA Stimulation Compared With T Percentages In Subject P .R .

PHA Stimulation + SEM Day 1 2 3 4 5

29923 _+ 942 35809 + 3093 31060 _+ 6777 35063 _+ 1691 23844 + 3124

T Percent

74 71 74 71 72

Correlation coefficient r = -0 .38 T and B Surface Markers On Frozen-Thawed Cells The percentages of T and B surface markers were determined on fresh as well as preserved cells . The results of the six subjects were averaged for each day and are shown in Table 3 as the mean + standard error of the mean . Comparisons of the percentages of the surface markers between fresh and frozen-thawed cells in each subject were made directly by paired-t analysis . No significant differences were found in any lymphocyte parameter . Variability of Lymphocyte Parameters In Fresh Cells.Compared To Frozen-Thawed Cells The average standard deviations of the SRBC rosette, AggIg, receptor, SIg and C-receptor percentages and PHA responses of the fresh and frozen-thawed

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No significant difference in variability becells are compared on Table 4 . tween fresh and frozen-thawed lymphocytes was noted in any parameter. Response Of Frozen-Thawed Cells To PHA Naximal PHA responsiveness of the lymphocytes was significantly enhanced in 4 out of 6 subjects, unchanged in 1, and depressed in 1 by the freeze-thaw procedure (Fig . 1) . Taken as a group increased responsiveness was seen follow ing the freeze-thaw procedure (p X0 .02) . The average increase in PHA responThe one siveness in the four subjects were 45, 59, 80 and 86% respectively . subject who showed a decrease had a 21% decline . Except for 1 subject, KR, who showed no change in PHA response on Day 1, the increase in response was noted in each of the five consecutive days . Discussion In the present study, we measured the daily variations in a number of widely used lymphocyte parameters . We took care to exclude all known factors which affect lymphocyte function such as smoking (14), exercise (15), steroids (16,17), serum catecholamine levels (12,18) and circadian hormonal changes (19). Cigarette smokers have higher leukocyte counts, neutrophils and monocyte percentages than nonsmokers (14) . Physical activity results in a marked increase in circulating lymphocytes with a fall in the proportion of SRBC rosette forming cells and a corresponding increase of cells with receptors for C3, IgG-Fc and surface immunoglobulin (15) . Oral and intravenous steroid administration produces a profound lymphopenia within 4 to 6 hours with a proportionately The administration of epinegreater decline in circulating T cells (16, 17) . phrine produces an increase in circulating lymphocytes (18) . We have shown that there are also a relative decrease in T cells, an increase in cells with AggIg-SIg and complement receptors, and an increase in cells with both SRBC and AggIg receptors (12) . A circadian rhythm in lymphocyte transformability to PHA has been found which coincided directly with the cortisol level (19) . To minimize these physiological variations, we studied only young male volunteers in good health who were nonsmokers and were taking no medications . Blood was drawn at a fixed time each morning after the subjects were adequately rested . Interobserver errors were reduced by having each person perform each set of investigations . 1. The variations of T lymphocytes We reached the following conclusions : as measured by the SRBC rosette assay were much greater when the total T conThe reason for this centrations were compared rather than the T percentages . is not certain. It has been well recognized that both absolute counts and lymphocyte percentages vary over a wide range in a normal population . A standard text reports that in a normal adult population, the lymphocyte count Such variranges between 1000 to 4000 per cu mm in the peripheral blood (20) . ations may in large part be attributable to the inaccuracy of the methods of assessing white cell counts and differentials (20) . Therefore, in studies. comparing the values of T cells in a single individual under different conditions, the percentages may reveal more subtle changes than the absolute concentrations . 2 . The daily variations of B cell percentages and PHA response were Variations of PHA reconsiderable in spite of the controlled circumstances . sponse were not related " to variations of T and B cell ratios . Consequently, comparing samples taken from the same individual under different conditions may not be meaningful unless the ranges of normal values are also taken into consideration . Ranges of normal values would have to :be established within each laboratory . Values falling outside this range may then be considered "abnormal" . 3 . No significant difference was noted in T and B percentages between This corroborates the findings of the fresh and frozen-thawed lymphocytes .

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others (6,7,21) . However, a significant enhancement of PHA responsiveness was observed after the freeze-thaw procedure . This was not due to a change in T and B cell ratios . Our data indicated that while the membrane markers of Afferent samples of fresh and frozen lymphocytes may be directly compared, direct comparison of PHA responsiveness cannot be made between fresh and frozen-thawed cells . There'is no total agreement on this point in the literature (7,22) . The present study differs in design in that instead of taking random samples from individuals, serial samples were taken . In this way, daily variations in PHA responsiveness were taken into account . In this study, cryopreservation was also used to try to reduce as much as possible the effect of the small and unavoidable changes in the composition of media and reagents . The freeze-thaw technique allowed us to thaw on the same day lymphocytes from a single donor which had been collected over five consecutive days and studied them under more uniform conditions with the same batch of reagents . We postulated that, had the technical variations been foremost in determining the day to day variability of our results, this would be reduced in the frozen-thawed lymphocytes which had been studied under more uniform conditions . Since the variability in the T and B percentages and responsiveness to PHA were almost identical in both the fresh and preserved lymphocytes (Table 4), we concluded that the technical factors were only in small part responsible for the variability of our results . Platz, et al (24) studied lymphocyte transformation to PHA in two normal individuals 39 times over an 18 months' period and found also wide day-to-day variation, but they felt that the variation in response could solely be accounted for by technical factors. Acknowledgments We wish to thank Ms . Edith Mehlworm and Ms . Evelyn Tackels for their excellent secretarial skills . REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 .

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J .L . Bernheim, Inserm, p . 147, Paris (1973) . N . Wood, H . Bash ,J . Greally, D .B . Amos, and E .J . Yunis, Tissue Antigens 62 27 .(1972) . M.A .S . Jewett, J .A . Hansen, and B . Dupont, Manual of Clinical Immunology ., p . 833 Am . Soc . Microbiol ., Washington (197-6- T P . Platz, T . Fog, N . Morling, A . Suejgaaxd, G . Sonderstrup, L .P . Ryder, M . Thomsen and C . Jersild, Acta Path . Microbiol . Scand . Sect . C, 84 501510 (1976) .