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Analysis of T cell subsets in normal adults
Journal of Immunological Methods, 98 (1987) 53-56
53
Elsevier JIM 04267
Analysis of T cell subsets in normal adults Comparison of whole blood lysis technique to Ficoll-Hypaque separation by flow cytometry P a o l o R e n z i * and Leo C. Ginns ** Pulmonary Unit, General Medical Services, Massachusetts General Hospital, and the Department of Medicine, Harvard Medical School Boston, MA, U.S.A. (Received 10 November 1986, accepted 26 November 1986)
Analysis of peripheral blood T cells and T cell subsets by immunofluorescence and flow cytometry is employed commonly in studies of normal individuals and in certain disease states. Methods for separating T cells from granulocytes and erythrocytes vary. We have sought to determine whether or not such procedures alter the composition of cells being analyzed. In this communication, we report that, in normal individuals, Ficoll-Hypaque separation of lymphocytes selectively decreases the OKT8 ÷ subset compared to the whole blood lysis technique (P < 0.01). The decrease in the OKT8 ÷ subset results in a significant increase (P < 0.01) in the OKT4/OKT8 ratio. This disparity appears to be due to a selective loss of OKT8 + cells during centrifugation over Ficoll-Hypaque. With the Ficoll-Hypaque method, we have also found that the fixation of lymphocytes reacted with monoclonal antibodies is less stable than cells prepared by the whole blood lysis method. The interpretation of measurements of T cell subsets is influenced by the method by which these cells are isolated. Key words: T lymphocyte; Monoclonal antibody; Flow cytometry; Ficoll-Hypaque; Whole blood
Introduction
Understanding the role of T cells and their subsets in disease states depends upon the ability to enumerate them accurately (Godard, 1981; Ginns, 1982a,b; Kornfeld, 1982). The advent of monoclonal antibodies reacting with specific cell surface antigens has facilitated these studies. C o u Correspondence to: L.C. Ginns, Pulmonary Unit, General Medical Services, Massachusetts General Hospital, 77 Fruit Street, Boston, MA 02114, U.S.A. * Work done during tenure of a research fellowship program NIH Training, Grant no. HL07354. ** Supported in part by the National Institute of Environmental Health Sciences, NIH Grant IROIESO330-2-02A1.
pied with flow cytometry in an immunofluorescent assay, enriched populations of cells can be rapidly and reproducibly analyzed (Reinherz, 1979; Ip, 1981; Miller, 1982). The validity of these results may depend, however, upon the method chosen to obtain the lymphocytes (BSyum, 1974; Berger, 1979; Hoffman, 1980; Hjorth, 1981). In our laboratory, no attempt is made to separate lymphocytes from other cells. Instead, the whole blood buffy coat is reacted with monoclonal antibodies. In other laboratories, lymphocytes are separated prior to their analysis. A commonly used technique for purification of monoclonal cells is that of Ficoll-Hypaque separation. This method relies upon differences in cell density in order to
0022-1759/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
54 separate lymphocytes from other cells. In this study, we compared the analysis of lymphocytes obtained by differing techniques in order to see whether the method of preparation alters results.
The bully coat was diluted with phosphatebuffered saline solution (PBS pH 7.4) to obtain a total of 4 ml. The cells were gently layered on top of an equal volume of lymphocyte separation medium (Bionetics) in 15 ml conical centrifuge tubes (Coming). The tubes were spun in the IEC centrifuge at 750 × g at room temperature for 45 rain. The mononuclear cells at the Ficoll interface were retrieved, washed twice in 10 ml of PBS, counted and resuspended at a concentration of 14 × 106 cells/ml in RPMI 1640 medium (Gibco).
min at room temperature, washed, and diluted to a final volume of 1 ml in PBS. The antibody treated cells were analyzed on a Spectrum III automated flow cytometer (Ortho Diagnostic Systems) (Hoffman, 1980). Scatter and fluorescence were standardized with an unstained and OKT3 ÷ preparation respectively (fluorescence peak = channel 70). Lymphocytes were distinguished from monocytes and neutrophils on the basis of their forward versus right angle light scatter. An electronic two-dimensional analysis window was set on the 'lymphocyte cluster', and its fluorescence determined as a 256 channel histogram in linear fluorescence units. The relative purity of the 'lymphocyte cluster' was determined by reacting cells with the monoclonal antibody OKM5 as described above. This antibody recognizes a determinant on human peripheral blood monocytes and granulocytes and is unreactive with lymphocytes (Breard, 1980). Generally, less than 5% of the cells within the 'lymphocytes cluster' reacted with this antibody. An average of 10008000 cells per sample were analyzed. Background staining was obtained by using a 0.07 ml aliquot of cells treated in the same way as above except that these cells were not incubated with an unconjugated antibody. The percentage of the negative control cells falling in the positive region was subtracted from the test sample to give the net percentage of positive cells.
Immunofluorescence staining
Conservation of fixed cells
Staining of buffy coat (Hoffman, 1980) and Ficoll-Hypaque prepared cells (B~Syum, 1974) were done simultaneously in an indirect immunofluorescent assay. Monoclonal antibodies used were OKT3 ÷, OKT4 +, OKT6 +, OKT8 + and OKIa, (Ortho Pharmaceuticals, Raritan, N J). Briefly, 0.07 ml of the cell suspension was incubated at 4°C with 0.01 ml of the different antibodies. The buffy coat prepared cells were treated twice with 2 ml of buffered ammonium chloride (pH 7.3) to lyse the erythrocytes and washed once in 2 ml of PBS. The Ficoll-Hypaque prepared cells were washed three times in 2 ml of PBS. The cells were then reacted with 0.05 ml of a 1 : 60 dilution of fluorescein-conjugated goat anti-mouse I g G + IgM antibody (Tago) at 4°C for 30 rain. Cells were fixed with 2.0% paraformaldehyde in PBS (pH 7.4) for 10
In five normal subjects, staining of buffy coat and Ficoll-Hypaque separated cells was done in duplicate. Half the cells were studied immediately, the other half was stored at 4 ° C and studied 72 h later.
Materials and methods
20-40 ml of venous blood were collected from 16 normal volunteers, 6 smokers and 10 nonsmokers, 9 males and 7 females, ages 24-47 (mean 30 years old). The blood remained at room temperature no more than 18 h in tubes containing acid-citrated dextrose solution. Following centrifugation of the blood on an IEC clinical centrifuge at room temperature for 15 min, the buffy coat was removed.
Ficoll-Hypaque separation
Statistical analysis Statistical analysis was performed using the paired and unpaired Student's t-test, a significant value having a P < 0.05.
Results
Table I shows the percentage of lymphocyte subsets obtained either by directly staining the buffy coat or by staining the cells after separation
55 TABLE I PERCENTAGE OF LYMPHOCYTESUBSETS IN WHOLE BLOOD BUFFY COAT OR AFTER SEPARATIONOVER FICOLL-HYPAQUE
TABLE II
16 patients studied except for OKIa (5). OKT3÷, OKT4+, OKT6+, OKT8+, OKIa + refers to reactivity of the respective monoclonal antibodies with antigens on the lymphocytemembranes.
Five volunteers studied.
OKT3÷ OKT4÷ OKT6+ OKT8÷ OKIa÷ T4/T8 ratio Buf~ coat Fic~lH~ue
71.6 43.6 0.3 26.1 8.2 1.8 -+7.7 -+8.2 -+0.4 -+5.9 -+2.7 -+0.6 70.0 42.6 0.8 16.8" 5.0 2.8* -+10 -+8.1 -+1.1 -+5.4 -+2.1 -+1.3
* P < 0.01.
on Ficoll-Hypaque. There is a significant decrease in the percentage of OKT8 ÷ cells after Ficoll-Hypaque separation. In every blood tested, the percentage of OKT8 + cells was lower by the FicollHypaque method compared to the whole blood buffy coat method. Since there was no change in the OKT4 + cells retrieved by both methods this produced a significant difference in the T 4 / T 8 ratios. These ratios are 2.8 and 1.8 for the FicollHypaque and whole blood technique respectively. The Ficoll-Hypaque separated lymphocytes also had lower O K I a markers, but this was not statistically significant. There was also no difference in the results obtained whether the volunteers were smokers or non-smokers. The percentage of OKT8 ÷ cells from the buffy coat were 2 6 . 5 _ 8% and 25.8 + 4% for the smokers and non-smokers respectively. The percentage of OKT8 ÷ cells from Ficoll-Hypaque separation were 17.8 + 5% and 16.3 + 6% for smokers and non-smokers. The cells obtained by the Ficoll-Hypaque method did not remain stable after fixation. After 72 h there was a significant increase in the background fluorescence for cells purified by the Ficoll-Hypaque method. The background increased from 4 _ 1.1 to 9.4 + 1.7 ( P < 0.05). At the same time, as shown in Table II, there was a significant decrease in the percentage of OKT3 +, OKT4 ÷ and OKT8 ÷ cells. By waiting 72 h before analyzing the Ficoll-Hypaque separated cells, not only were the OKT8 ÷ cells decreased, but the OKT3 ÷ and OKT4 ÷ cells were also decreased.
PERCENTAGE OF LYMPHOCYTE SUBSETS AFTER SEPARATION OVER FICOLL-HYPAQUE,AT 0 to 72 h AFTER STAINING
Time OKT3 OKT4 OKT60KT8 T4/T8 Ficoll0h Hypaque
61.6 +8.0
43.1 q-4
0.2 +0.2
17.5 2.5 _+2.6 -+0.2
Ficoll72 h Hypaque
56.4* 40.0 * 0.8 14.2 * 2.8 -+5.7 -+3.1 -+0.3 +2.7 -+0.4
• P < 0.01 buffy coat vs. Ficoll-Hypaqueat 72 h. There was no change, however, in any of the results obtained for each T cell subset by the buffy coat method, whether they were studied immediately or 72 h later. Discussion
In this study we have found that, in normal individuals, Ficoll-Hypaque separation of lymphocytes selectively decreases the OKT8 ÷ subset compared to the whole blood lysis technique. The decrease in the OKT8 ÷ subset results in a significant increase in the O K T 4 / O K T 8 ratio. In addition, separation of lymphocytes over FicollHypaque reduces the stability of the staining and fixation procedure. We suggest that cells obtained by Ficoll-Hypaque separation should be analyzed as soon as possible to avoid an increase in background and a decrease in the OKT3 ÷, OKT4 ÷ and OKT8 + ceils. Ficoll-Hypaque separation of lymphocytes from whole blood is known to modify certain subsets and properties of lymphocytes. In rabbits, an increase in B cells and a decrease in T cells occurs by this method at 4 ° C and at room temperature (Srinivasa Upuda, 1980). In these animals, there is selective binding of T cells to erythrocytes during separation and precipitation to the bottom of the centrifuge tube. Human mononuclear cells retrieved by elutriation compared to those obtained by Ficoll-Hypaque separation are 130% more responsive to streptokinase and streptodornase (Berger, 1979). Leu-7 + and L e u - l l + cells with high natural killer activity are increased in the mononuclear cells retrieved by the Ficoll-Hypaque method (DePaoli, 1984). Our results show a consistent selec-
56 tive decrease in the percentage of OKT8 ÷ cells and thus an increase in the T 4 / T 8 ratio of lymphocytes obtained by this method. Thorntwaite (1984) found no difference in the T 4 / T 8 ratio between both methods, but the whole blood was studied by flow cytometry and the Ficoll-Hypaque prepared lymphocytes were studied by manual counting under microscopy. Nicholson (1984) concluded that there were no differences in subsets by both methods but only five patients were studied and the calculated T 4 / T 8 ratio from their results is 1.8 for the whole blood lysis method and 2.3 for the Ficoll-Hypaque. We believe our results are not in conflict with theirs. The differences found in the OKT8 + populations and the T 4 / T 8 ratios could be explained in two ways: either a selective increase in the OKT8 + population by the whole blood lysis method or a selective decrease in the OKT8 + cells by the Ficoll-Hypaque method. DePaoli (1984) has shown a selective loss of OKT8 + cells at the bottom of their centrifuge tubes by Ficoll-Hypaque separation. Iwatani (1982) found that lysis of erythrocytes after separation of lymphocytes over Ficoll metrizoate decreased the OKT8 + population and increased the T 4 / T 8 ratio. Both these results support the hypothesis that there is a selective loss of the OKT8 + population during Ficoll-Hypaque separation. The separation of mononuclear cells from granulocytes and erythrocytes during Ficoll-Hypaque centrifugation depends upon the density of the cells concerned, the density of the Ficoll-Hypaque solution and the ability of red blood cells to form aggregates and precipitate at the bottom of the tube. Two alternatives might explain the selective loss of OKT8 + cells: the OKT8 + cells might have a higher density than other T cells, or OKT8 + cells might form aggregates with red blood cells and precipitate during centrifugation. Speculation favors the second hypothesis because it has already been shown that T cells can form aggregates with red blood cells in rabbits (Srinivasa, 1982). In conclusion, our results show a selective decrease in the OKT8 + population of lymphocytes and an increase in the T 4 / T 8 ratio by the FicollHypaque method of separating mononuclear cells from whole blood. The fixation of lymphocytes isolated by this technique also seems less stable.
The T 4 / T 8 ratio is altered in several disease states (Ginns, 1982a, b; Kornfeld, 1982; Jacobs, 1985). When studying the T 4 / T 8 ratio, careful attention must be paid to whether the lymphocytes have been obtained by the Ficoll-Hypaque or whole blood lysis method.
Acknowledgements The authors thank Gideon Goldstein for his advice and the Ortho Pharmaceutical Corporation for providing monoclonal antibodies and Dr. Homayoun Kazemi for helpful suggestions.
References Berger, C.L. and Edelson, R.L. (1979) J. Invest. Dermatol. 73, 231. BSyum, A. (1974) Tissue Antigens 4, 269. Breard, J., Reinherz, E.L. Kung, P.C., Goldstein, G. and Schlossman, S. (1980) J. Immunol. 124, 1943. DePaoli, P., Reitano, M., Battistin, S., Castiglia, C. and Santini, G. (1984) J. Immunol. Methods 72, 349. Ginns, L.C., Goldenheim, P.D., Burton, R.C., Colvin, R.B., Mitler, L.G., Goldstein, G., Hurwitz, C. and Kazemi, H. (1982a) Clin. Immunol. Immunopathol. 25, 11. Ginns, L.C., Goldenheim, P.D., Miller, L.G., Burton, R.C., Gillick, L., Colvin, R.B., Goldstein, G., Kung, P.C., Hurwitz, C. and Kazemi, H. (1982b) Am. Rev. Respir. Dis. 126, 265. Hjorth, R., Jonsson, A. and Vretblad, P. (1981) J. Immunol. Methods 43, 95. Hoffman, R.A., Kung, P.C., Hansen, W.P. and Goldstein, G. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 4914. Ip, S.H., Ritterhaus, C.W., Healy, K.W., Struzziero, L.C., Hoffman, R.A. and Hansen, P.W. (1982) Clin. Chem. 28, 1905. Iwatani, Y., Amino, N., Moil, H., Asani, S., Ina, K., Ennyw, K. and Miyai, K. (1982) J. Immunol. Methods 54, 31. Jacobs, R.F., Marmer, D.J., Bolk, R.A. and Bradsher, R.W. (1985) Chest 88, 579. Kornfeld, H., Vande Stowe, R.A., Lange, M., Mohan Reddy, M. and Grieco, M. (1982) N. Engl. J. Med. 307, 729. Miller, L.G., Goldstein, G., Murphy, M. and Ginns, L.C. (1982) Chest 82, 526. Nicholson, J.K.A., Jones, B.M., Gross, G.D. and McDougal, J.S. (1984) J. Immunol. Methods 73, 29. Reinherz, E.L., Kung, P.C., Goldstein, G. and Schlossman, S.F. (1979) J. Immunol. 123, 1312. Schroff, R.W., Foon, K.A., Billing, R.J. and Fahney, J.L. (1982) Blood 59, 207. Srinivasa Upuda, T.N., Chow, C.T., Cavaillon, J.M., Cinader, B. and Dubiski, S. (1980) J. Immunol. Methods 38, 201. Thorntwaite, T., Seckinger, D., Sugarbaker, E.V., Rosenthal, P.K. and Vazquez, D.A. (1984) Am. J. Clin. Pathol. 82, 48.
53
Elsevier JIM 04267
Analysis of T cell subsets in normal adults Comparison of whole blood lysis technique to Ficoll-Hypaque separation by flow cytometry P a o l o R e n z i * and Leo C. Ginns ** Pulmonary Unit, General Medical Services, Massachusetts General Hospital, and the Department of Medicine, Harvard Medical School Boston, MA, U.S.A. (Received 10 November 1986, accepted 26 November 1986)
Analysis of peripheral blood T cells and T cell subsets by immunofluorescence and flow cytometry is employed commonly in studies of normal individuals and in certain disease states. Methods for separating T cells from granulocytes and erythrocytes vary. We have sought to determine whether or not such procedures alter the composition of cells being analyzed. In this communication, we report that, in normal individuals, Ficoll-Hypaque separation of lymphocytes selectively decreases the OKT8 ÷ subset compared to the whole blood lysis technique (P < 0.01). The decrease in the OKT8 ÷ subset results in a significant increase (P < 0.01) in the OKT4/OKT8 ratio. This disparity appears to be due to a selective loss of OKT8 + cells during centrifugation over Ficoll-Hypaque. With the Ficoll-Hypaque method, we have also found that the fixation of lymphocytes reacted with monoclonal antibodies is less stable than cells prepared by the whole blood lysis method. The interpretation of measurements of T cell subsets is influenced by the method by which these cells are isolated. Key words: T lymphocyte; Monoclonal antibody; Flow cytometry; Ficoll-Hypaque; Whole blood
Introduction
Understanding the role of T cells and their subsets in disease states depends upon the ability to enumerate them accurately (Godard, 1981; Ginns, 1982a,b; Kornfeld, 1982). The advent of monoclonal antibodies reacting with specific cell surface antigens has facilitated these studies. C o u Correspondence to: L.C. Ginns, Pulmonary Unit, General Medical Services, Massachusetts General Hospital, 77 Fruit Street, Boston, MA 02114, U.S.A. * Work done during tenure of a research fellowship program NIH Training, Grant no. HL07354. ** Supported in part by the National Institute of Environmental Health Sciences, NIH Grant IROIESO330-2-02A1.
pied with flow cytometry in an immunofluorescent assay, enriched populations of cells can be rapidly and reproducibly analyzed (Reinherz, 1979; Ip, 1981; Miller, 1982). The validity of these results may depend, however, upon the method chosen to obtain the lymphocytes (BSyum, 1974; Berger, 1979; Hoffman, 1980; Hjorth, 1981). In our laboratory, no attempt is made to separate lymphocytes from other cells. Instead, the whole blood buffy coat is reacted with monoclonal antibodies. In other laboratories, lymphocytes are separated prior to their analysis. A commonly used technique for purification of monoclonal cells is that of Ficoll-Hypaque separation. This method relies upon differences in cell density in order to
0022-1759/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
54 separate lymphocytes from other cells. In this study, we compared the analysis of lymphocytes obtained by differing techniques in order to see whether the method of preparation alters results.
The bully coat was diluted with phosphatebuffered saline solution (PBS pH 7.4) to obtain a total of 4 ml. The cells were gently layered on top of an equal volume of lymphocyte separation medium (Bionetics) in 15 ml conical centrifuge tubes (Coming). The tubes were spun in the IEC centrifuge at 750 × g at room temperature for 45 rain. The mononuclear cells at the Ficoll interface were retrieved, washed twice in 10 ml of PBS, counted and resuspended at a concentration of 14 × 106 cells/ml in RPMI 1640 medium (Gibco).
min at room temperature, washed, and diluted to a final volume of 1 ml in PBS. The antibody treated cells were analyzed on a Spectrum III automated flow cytometer (Ortho Diagnostic Systems) (Hoffman, 1980). Scatter and fluorescence were standardized with an unstained and OKT3 ÷ preparation respectively (fluorescence peak = channel 70). Lymphocytes were distinguished from monocytes and neutrophils on the basis of their forward versus right angle light scatter. An electronic two-dimensional analysis window was set on the 'lymphocyte cluster', and its fluorescence determined as a 256 channel histogram in linear fluorescence units. The relative purity of the 'lymphocyte cluster' was determined by reacting cells with the monoclonal antibody OKM5 as described above. This antibody recognizes a determinant on human peripheral blood monocytes and granulocytes and is unreactive with lymphocytes (Breard, 1980). Generally, less than 5% of the cells within the 'lymphocytes cluster' reacted with this antibody. An average of 10008000 cells per sample were analyzed. Background staining was obtained by using a 0.07 ml aliquot of cells treated in the same way as above except that these cells were not incubated with an unconjugated antibody. The percentage of the negative control cells falling in the positive region was subtracted from the test sample to give the net percentage of positive cells.
Immunofluorescence staining
Conservation of fixed cells
Staining of buffy coat (Hoffman, 1980) and Ficoll-Hypaque prepared cells (B~Syum, 1974) were done simultaneously in an indirect immunofluorescent assay. Monoclonal antibodies used were OKT3 ÷, OKT4 +, OKT6 +, OKT8 + and OKIa, (Ortho Pharmaceuticals, Raritan, N J). Briefly, 0.07 ml of the cell suspension was incubated at 4°C with 0.01 ml of the different antibodies. The buffy coat prepared cells were treated twice with 2 ml of buffered ammonium chloride (pH 7.3) to lyse the erythrocytes and washed once in 2 ml of PBS. The Ficoll-Hypaque prepared cells were washed three times in 2 ml of PBS. The cells were then reacted with 0.05 ml of a 1 : 60 dilution of fluorescein-conjugated goat anti-mouse I g G + IgM antibody (Tago) at 4°C for 30 rain. Cells were fixed with 2.0% paraformaldehyde in PBS (pH 7.4) for 10
In five normal subjects, staining of buffy coat and Ficoll-Hypaque separated cells was done in duplicate. Half the cells were studied immediately, the other half was stored at 4 ° C and studied 72 h later.
Materials and methods
20-40 ml of venous blood were collected from 16 normal volunteers, 6 smokers and 10 nonsmokers, 9 males and 7 females, ages 24-47 (mean 30 years old). The blood remained at room temperature no more than 18 h in tubes containing acid-citrated dextrose solution. Following centrifugation of the blood on an IEC clinical centrifuge at room temperature for 15 min, the buffy coat was removed.
Ficoll-Hypaque separation
Statistical analysis Statistical analysis was performed using the paired and unpaired Student's t-test, a significant value having a P < 0.05.
Results
Table I shows the percentage of lymphocyte subsets obtained either by directly staining the buffy coat or by staining the cells after separation
55 TABLE I PERCENTAGE OF LYMPHOCYTESUBSETS IN WHOLE BLOOD BUFFY COAT OR AFTER SEPARATIONOVER FICOLL-HYPAQUE
TABLE II
16 patients studied except for OKIa (5). OKT3÷, OKT4+, OKT6+, OKT8+, OKIa + refers to reactivity of the respective monoclonal antibodies with antigens on the lymphocytemembranes.
Five volunteers studied.
OKT3÷ OKT4÷ OKT6+ OKT8÷ OKIa÷ T4/T8 ratio Buf~ coat Fic~lH~ue
71.6 43.6 0.3 26.1 8.2 1.8 -+7.7 -+8.2 -+0.4 -+5.9 -+2.7 -+0.6 70.0 42.6 0.8 16.8" 5.0 2.8* -+10 -+8.1 -+1.1 -+5.4 -+2.1 -+1.3
* P < 0.01.
on Ficoll-Hypaque. There is a significant decrease in the percentage of OKT8 ÷ cells after Ficoll-Hypaque separation. In every blood tested, the percentage of OKT8 + cells was lower by the FicollHypaque method compared to the whole blood buffy coat method. Since there was no change in the OKT4 + cells retrieved by both methods this produced a significant difference in the T 4 / T 8 ratios. These ratios are 2.8 and 1.8 for the FicollHypaque and whole blood technique respectively. The Ficoll-Hypaque separated lymphocytes also had lower O K I a markers, but this was not statistically significant. There was also no difference in the results obtained whether the volunteers were smokers or non-smokers. The percentage of OKT8 ÷ cells from the buffy coat were 2 6 . 5 _ 8% and 25.8 + 4% for the smokers and non-smokers respectively. The percentage of OKT8 ÷ cells from Ficoll-Hypaque separation were 17.8 + 5% and 16.3 + 6% for smokers and non-smokers. The cells obtained by the Ficoll-Hypaque method did not remain stable after fixation. After 72 h there was a significant increase in the background fluorescence for cells purified by the Ficoll-Hypaque method. The background increased from 4 _ 1.1 to 9.4 + 1.7 ( P < 0.05). At the same time, as shown in Table II, there was a significant decrease in the percentage of OKT3 +, OKT4 ÷ and OKT8 ÷ cells. By waiting 72 h before analyzing the Ficoll-Hypaque separated cells, not only were the OKT8 ÷ cells decreased, but the OKT3 ÷ and OKT4 ÷ cells were also decreased.
PERCENTAGE OF LYMPHOCYTE SUBSETS AFTER SEPARATION OVER FICOLL-HYPAQUE,AT 0 to 72 h AFTER STAINING
Time OKT3 OKT4 OKT60KT8 T4/T8 Ficoll0h Hypaque
61.6 +8.0
43.1 q-4
0.2 +0.2
17.5 2.5 _+2.6 -+0.2
Ficoll72 h Hypaque
56.4* 40.0 * 0.8 14.2 * 2.8 -+5.7 -+3.1 -+0.3 +2.7 -+0.4
• P < 0.01 buffy coat vs. Ficoll-Hypaqueat 72 h. There was no change, however, in any of the results obtained for each T cell subset by the buffy coat method, whether they were studied immediately or 72 h later. Discussion
In this study we have found that, in normal individuals, Ficoll-Hypaque separation of lymphocytes selectively decreases the OKT8 ÷ subset compared to the whole blood lysis technique. The decrease in the OKT8 ÷ subset results in a significant increase in the O K T 4 / O K T 8 ratio. In addition, separation of lymphocytes over FicollHypaque reduces the stability of the staining and fixation procedure. We suggest that cells obtained by Ficoll-Hypaque separation should be analyzed as soon as possible to avoid an increase in background and a decrease in the OKT3 ÷, OKT4 ÷ and OKT8 + ceils. Ficoll-Hypaque separation of lymphocytes from whole blood is known to modify certain subsets and properties of lymphocytes. In rabbits, an increase in B cells and a decrease in T cells occurs by this method at 4 ° C and at room temperature (Srinivasa Upuda, 1980). In these animals, there is selective binding of T cells to erythrocytes during separation and precipitation to the bottom of the centrifuge tube. Human mononuclear cells retrieved by elutriation compared to those obtained by Ficoll-Hypaque separation are 130% more responsive to streptokinase and streptodornase (Berger, 1979). Leu-7 + and L e u - l l + cells with high natural killer activity are increased in the mononuclear cells retrieved by the Ficoll-Hypaque method (DePaoli, 1984). Our results show a consistent selec-
56 tive decrease in the percentage of OKT8 ÷ cells and thus an increase in the T 4 / T 8 ratio of lymphocytes obtained by this method. Thorntwaite (1984) found no difference in the T 4 / T 8 ratio between both methods, but the whole blood was studied by flow cytometry and the Ficoll-Hypaque prepared lymphocytes were studied by manual counting under microscopy. Nicholson (1984) concluded that there were no differences in subsets by both methods but only five patients were studied and the calculated T 4 / T 8 ratio from their results is 1.8 for the whole blood lysis method and 2.3 for the Ficoll-Hypaque. We believe our results are not in conflict with theirs. The differences found in the OKT8 + populations and the T 4 / T 8 ratios could be explained in two ways: either a selective increase in the OKT8 + population by the whole blood lysis method or a selective decrease in the OKT8 + cells by the Ficoll-Hypaque method. DePaoli (1984) has shown a selective loss of OKT8 + cells at the bottom of their centrifuge tubes by Ficoll-Hypaque separation. Iwatani (1982) found that lysis of erythrocytes after separation of lymphocytes over Ficoll metrizoate decreased the OKT8 + population and increased the T 4 / T 8 ratio. Both these results support the hypothesis that there is a selective loss of the OKT8 + population during Ficoll-Hypaque separation. The separation of mononuclear cells from granulocytes and erythrocytes during Ficoll-Hypaque centrifugation depends upon the density of the cells concerned, the density of the Ficoll-Hypaque solution and the ability of red blood cells to form aggregates and precipitate at the bottom of the tube. Two alternatives might explain the selective loss of OKT8 + cells: the OKT8 + cells might have a higher density than other T cells, or OKT8 + cells might form aggregates with red blood cells and precipitate during centrifugation. Speculation favors the second hypothesis because it has already been shown that T cells can form aggregates with red blood cells in rabbits (Srinivasa, 1982). In conclusion, our results show a selective decrease in the OKT8 + population of lymphocytes and an increase in the T 4 / T 8 ratio by the FicollHypaque method of separating mononuclear cells from whole blood. The fixation of lymphocytes isolated by this technique also seems less stable.
The T 4 / T 8 ratio is altered in several disease states (Ginns, 1982a, b; Kornfeld, 1982; Jacobs, 1985). When studying the T 4 / T 8 ratio, careful attention must be paid to whether the lymphocytes have been obtained by the Ficoll-Hypaque or whole blood lysis method.
Acknowledgements The authors thank Gideon Goldstein for his advice and the Ortho Pharmaceutical Corporation for providing monoclonal antibodies and Dr. Homayoun Kazemi for helpful suggestions.
References Berger, C.L. and Edelson, R.L. (1979) J. Invest. Dermatol. 73, 231. BSyum, A. (1974) Tissue Antigens 4, 269. Breard, J., Reinherz, E.L. Kung, P.C., Goldstein, G. and Schlossman, S. (1980) J. Immunol. 124, 1943. DePaoli, P., Reitano, M., Battistin, S., Castiglia, C. and Santini, G. (1984) J. Immunol. Methods 72, 349. Ginns, L.C., Goldenheim, P.D., Burton, R.C., Colvin, R.B., Mitler, L.G., Goldstein, G., Hurwitz, C. and Kazemi, H. (1982a) Clin. Immunol. Immunopathol. 25, 11. Ginns, L.C., Goldenheim, P.D., Miller, L.G., Burton, R.C., Gillick, L., Colvin, R.B., Goldstein, G., Kung, P.C., Hurwitz, C. and Kazemi, H. (1982b) Am. Rev. Respir. Dis. 126, 265. Hjorth, R., Jonsson, A. and Vretblad, P. (1981) J. Immunol. Methods 43, 95. Hoffman, R.A., Kung, P.C., Hansen, W.P. and Goldstein, G. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 4914. Ip, S.H., Ritterhaus, C.W., Healy, K.W., Struzziero, L.C., Hoffman, R.A. and Hansen, P.W. (1982) Clin. Chem. 28, 1905. Iwatani, Y., Amino, N., Moil, H., Asani, S., Ina, K., Ennyw, K. and Miyai, K. (1982) J. Immunol. Methods 54, 31. Jacobs, R.F., Marmer, D.J., Bolk, R.A. and Bradsher, R.W. (1985) Chest 88, 579. Kornfeld, H., Vande Stowe, R.A., Lange, M., Mohan Reddy, M. and Grieco, M. (1982) N. Engl. J. Med. 307, 729. Miller, L.G., Goldstein, G., Murphy, M. and Ginns, L.C. (1982) Chest 82, 526. Nicholson, J.K.A., Jones, B.M., Gross, G.D. and McDougal, J.S. (1984) J. Immunol. Methods 73, 29. Reinherz, E.L., Kung, P.C., Goldstein, G. and Schlossman, S.F. (1979) J. Immunol. 123, 1312. Schroff, R.W., Foon, K.A., Billing, R.J. and Fahney, J.L. (1982) Blood 59, 207. Srinivasa Upuda, T.N., Chow, C.T., Cavaillon, J.M., Cinader, B. and Dubiski, S. (1980) J. Immunol. Methods 38, 201. Thorntwaite, T., Seckinger, D., Sugarbaker, E.V., Rosenthal, P.K. and Vazquez, D.A. (1984) Am. J. Clin. Pathol. 82, 48.