Cell Tissue Res. 204, 147-153 (1979)
Cell and Tissue Research 9 by Springer-Verlag 1979
Scanning Electron Microscopy of Peripheral Blood Leukocytes of the Chicken Eberhard Burkhardt Institut fiir Veterinfir-Pathologieder Justus Liebig-Universit~it Giessen, Giessen, Bundesrepublik Deutschland
Summary.Polymorphonuclear leukocytes, e.g., neutrophilic granulocytes, were .enriched from heparinized blood by a Ficoll-step-gradient centrifugation procedure. Scanning electron microscopy (SEM) revealed a surface morphology of narrow ridge-like profiles and small ruffles with occasional microprocesses. Mononuclear leukocytes were isolated by centrifugation over a Ficoll-Metrizoat gradient. The lymphocytes showed varying numbers of microvilli of different length, size and shape. B lymphocytes, characterized by their capability of"sheep red blood cell (SRBC)-rosette formation", displayed a similar surface morphology. Completely smooth lymphocytes, described in the literature as T lymphocytes, could not be detected, although many lymphocytes with few microprocesses were observed. Thus, SEM is not a useful tool for distinguishing between B and T lymphocytes in the peripheral blood of chickens. Monocytes were characterized by prominent membrane-like ruffles, but in some cases they closely resembled granulocytes. An influence of the various separation media on the surface morphology of the isolated cells could not be detected when compared with cells isolated by the buffy-coat method. Key words: Granulocytes - Lymphocytes - Monocytes - Scanning electron microscopy - Chicken.
Efforts to distinguish B from T lymphocytes scanning electron microscopically (SEM) have been controversial. Some authors have described a clear-cut surface morphology of human and murine B and T cells (Polliack et al., 1973, 1975; Linthicum et al., 1974; van Ewijk and Brons, 1976; Polliack, 1977). B cells show a rough surface with many microvilli, while T cells reveal a relatively smooth cell surface. Other authors have been unable to confirm these findings (Alexander and Send offprint requests to: Dr. Eberhard Burkhardt, Institut for Veterin~ir-Pathologieder Justus Liebig-
Universit~it, Frankfurter StraBe 96, D-6300 Giessen, Federal Republic of Germany Acknowledgements: This study was supported by the SFB 47 of the Deutsche Forschungsgerneinschaft, Federal Republic of Germany. The author thanks Mrs. U. Zeller for photographical assistance
0302-766X/79/0204/0147/$01.40
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W e t z e l , 1975; B a u r et al., 1975; A l e x a n d e r et al., 1976; D a n t c h e v a n d B e l p o m m e , 1977). I n the c h i c k e n , A k i y a m a a n d K a t o (1974) a n d N a z e r i a n et al. (1976) f o u n d a r o u g h s u r f a c e o f cells d e r i v e d f r o m the b u r s a o f F a b r i c i u s , w h i l e cells f r o m the thymus showed a smooth surface without microprocesses. In contrast, Stinson and G l i c k (1978) o b s e r v e d the s m o o t h - c e l l t y p e to be the m o s t p r o m i n e n t , r e g a r d l e s s o f the o r i g i n o f the l y m p h o c y t e s . I n the p e r i p h e r a l b l o o d o f the c h i c k e n , A k i y a m a a n d K a t o (1974) o b s e r v e d t h a t a b o u t 50 % o f t h e l y m p h o c y t e s h a v e a r o u g h s u r f a c e morphology with many long microvilli. I n the p r e s e n t p a p e r the S E M - m o r p h o l o g y o f p e r i p h e r a l b l o o d l e u k o c y t e s o f the c h i c k e n , s e p a r a t e d b y d i f f e r e n t m e t h o d s for o b t a i n i n g s u b g r o u p s o f l e u k o c y t e s , is d e s c r i b e d . F u r t h e r m o r e , t h e s u r f a c e m o r p h o l o g y o f l y m p h o c y t e s w a s inv e s t i g a t e d in a n e f f o r t to d i s t i n g u i s h b e t w e e n T a n d B l y m p h o c y t e s .
Materials and Methods Animals and Blood. Heparinized (50 I.U./ml) blood was taken from the wing vein of 6 month-old White Leghorn chickens. Enrichment ofPolymorphonuclear Leukocytes. A Ficoll-gradient-step centrifugation method described by Noble and Cutts (1968) was used. Briefly, 5 ml of heparinized blood was layered over 9 ml of a 35 % Ficoll (MG 400000 ) solution in Hank's buffered salt solution (HBSS) and spun at 1000 g for 30 min. The band of white cells on the top of the gradient was washed once in HBSS and layered over a Ficoll-step gradient ranging from 35 % Ficoll solution in HBSS in the bottom layer to 14.5 % Ficoll solution in HBSS in the top layer. The cells were spun at 50g for 5min followed by 300g for 10min. After centrifugation polymorphonuclear leukocytes were found in the 24% and 18 % Ficoll-gradient steps, from which they were removed by use of a Pasteur pipette. Isolation ofMononuclear Leukocytes. The Ficoll-Metrizoat-gradient centrifugation method (Boyum, 1968; Coudert and Richard, 1975) was used. 5 ml of heparinized blood was layered over 2 ml FicollRonpacon | separation medium and spun at 600 g for 20min in a 12ml glass centrifuge tube. The isolated mononuclear leukocytes were found in a small opaque ring at the interface of the separation medium and plasma, from which they were removed with a Pasteur pipette. Isolation of Total White Blood Cells in a Buffy Coat. 10 ml of heparinized blood were spun at 1500 g for 10 min. The resulting buffy coat containing all white blood cells was removed from the underlying red cell column by means of a Pasteur pipette. Isolation of B Lymphoeytes by Sheep Red Blood Cell (SRBC)-Rosette Formation. For SRBC-rosette formation the method described by Ivanyi et al. (1976) was used. Briefly, 6 days after intravenous immunization with 3 % SRBC suspension in HBSS (1 ml/kg body weight), 30 ml of heparinized blood was taken from a chicken by vein puncture. The mononuclear leukocytes were isolated by FicollMetrizoat-gradient centrifugation. 0.1 ml of a washed 2 % SRBC suspension in HBSS was mixed with an equal volume of the isolated cell suspension (2 • l0 Tcells/ml) in glass centrifuge tubes with round bottoms and kept on ice for 5 min. The tubes were spun at 200 g for 7 min and kept on ice again for 30min. The cell pellets were gently resuspended, pooled and suspended in HBSS to a total volume of 5 ml, layered over 2 ml of FicoU-Ronpacon| and spun at 600 g for 20 min. Then the SRBC rosetteforming cells were found in the red cell column at the bottom of the centrifuge tube, while the nonrosette-forming mononuclear leukocytes were found together with a few SRBC in a small ring at the interface of the separation medium and overlying buffer. Both cell fractions were prepared for SEM. Preparation of Cells for SEM. Cells obtained by the various separation methods were washed twice (200 g, 5 min) in HBSS, resuspended in 1 ml HBSS and injected with a Pasteur pipette into 10 ml of 1.2 % glutaraldehyde in 0.1 M cacodylate buffer (pH = 7.3, osmolality = 300 mO smol) and fixed for 1 h. The
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cells were washed twice in 0.16M cacodylate buffer (pH = 7.3, osmolality = 300mOsmol) and postfixed in 1 ~ OsO4 in 0.1 M cacodylate buffer (pH = 7.3, osmolality = 225 mOsmol) for 1 h. The cells were dehydrated in a graded series of ethanol and in three fractions of absolute ethanol (minimum time of 10 min for each step). The cells were then processed through mixtures of ethanol/iso-amyl acetate 3 : 1, 1 : 1, I : 3 and through two fractions of pure iso-amyl acetate. Three drops of this cell suspension were brought onto small aluminium membrane dishes with a diameter o f about 10 mm and allowed to settle for 2 min. The cells were critical-point dried in CO 2 in a Balzers Union Critical Point Dryer, the aluminium dishes attached to aluminium stubs with a carbon-propanol glue and coated with gold in a Balzers Union Sputtering device. The preparations were examined with a Siemens Autoscan at 20 kV and a tilt angle of 45 ~ Photographs were taken with Agfapan Professional 25 film.
Light Microscopy. Giemsa-stained smears prepared from each cell suspension obtained by the various separation procedures were used as controls.
Fig. l a d . Scanning electron micrographs, a Aggregated granulocytes, x 4200. b Granulocyte, x 10,600. c Granulocyte with small microprocesses (arrows), x 10,500. d Monocyte with broad and prominent ruffles (arrows), x 12,600
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Fig. 2a-i. Lymphocytes showing microvilli of different number, length and shape. Highly villous forms: a x 11,000; b x 9600; e x 9800. Less villous forms: d • 8000; e x 10,600; f x 9700. Relatively smooth forms: g • 10,000; h • 9400; i x 9700
Results A m o n g Ficoll gradient-step and buffy coat-separated cells m a n y spherical cells with a diameter o f 5-6 ~tm were found, which tended to aggregate (Fig. 1 a). Their cell surface showed n a r r o w ridge-like profiles and small ruffles (Fig. l a-c) and occasionally a few microprocesses (Fig. lc, arrows). In Giemsa-stained smears these cells were identified as p o l y m o r p h o n u c l e a r leukocytes; m o s t o f them were heterophilic granulocytes. Some cells with a similar surface m o r p h o l o g y , but m o r e prominent, membrane-like ruffles (Fig. 1 d, arrows), were f o u n d in the buffy coat and in Ficoll-Metrizoat-separated cell suspensions. These cells were identified as monocytes in Giemsa-stained smears. Most o f the Ficoll-Metrizoat-separated cells were identified as lymphocytes in Giemsa-stained smears. They were 3-5 ktm in diameter and had a varying n u m b e r o f microvilli (Fig. 2a-i). Very few cells
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Fig. 3a and b. SRBC rosette-formingcells (B-lymphocytes)displayingdifferentsurface morphology. a x5400; b x5600
displayed rather long "spiky" microvilli (Fig. 2g), while most of them showed relatively short microvilli. Some of these were stud-like (Fig. 2e) or "coral"-shaped (Fig. 2 a), while others were finger-like (Fig. 2 b, c, d). There were large differences in the number of microvilli per cell. Cells with a highly villous (Fig. 2 a-c), a less villous (Fig. 2d-f) and a relatively smooth cell surface (Fig. 2g-i) were encountered. The SRBC rosette-forming cells displayed microvilli of varying numbers and shape and were attached to some spheroechinocytic erythrocytes (Fig. 3a, b). Some rosetteforming cells possessed a coral-like surface (Fig. 3 a), while others showed a highly villous cell surface characterized by finger-like microvilli (Fig. 3b). All SRBC rosette-forming cells were identified light microscopically as lymphocytes; they showed the same SEM-morphology as the lymphocytes not forming SRBC rosettes. Differences in surface morphology of the various subgroups of leukocytes after isolation with the buffy-coat method and with the gradient media were not observed.
Discussion
In the present study, the enriched polymorphonuclear leukocytes of the chicken were characterized by ridge-like profiles and small ruffles. Their surface morphology was similar to that of human neutrophilic granulocytes described by Polliack (1977), while human eosinophils and basophils reveal a different surface structure (Polliack, 1977). From the present studies, it could not be conclusively determined that heterophilic granulocytes differ in their surface morphology from eosinophils and basophils, because we were not able to separate these subgroups of granulocytes. A few cells with a similar structure of the surface, but with broader and more prominent ruffles were observed in Ficoll-Metrizoat gradient and in buffy coat-separated cell suspensions. These cells were characterized in Giemsa-
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stained smears as monocytes. The same SEM-surface morphology was described for monocytes in human blood by Polliack (1977). Sometimes, these cells were confused with polymorphonuclear leukocytes in the SEM. Lymphocytes showed a surface morphology different from that of the abovedescribed cell elements. They are characterized by varying numbers ofmicrovilli of different length and shape. However, a clear-cut surface morphology for two different subgroups of lymphocytes, as described in the chicken, for bursa and thymus cells by Akiyama and Kato (1974) and Nazerian et al. (1976), was not observed. These authors emphasized that bursal cells possess a villous surface. In our studies the cell surface morphology of peripheral blood lymphocytes varied continuously from highly villous, less villous, to relatively smooth forms. However, the percentage of B lymphocytes in chicken peripheral blood ranges from 5-15 ~, as has been indicated by the presence of surface immunoglobulin (Albini and Wick, 1973; Hoffmann-Fezer et al., 1973; Ivanyi and Hudson, 1978). SRBC rosetteforming cells, which were characterized as B lymphocytes by Ivanyi et al. (1976), also varied from highly villous (Fig. 3 b) forms to cells with fewer microvilli. Lymphocytes with a completely smooth cell surface and lacking microvilli, as described by Akiyama and Kato (1974), Nazerian et al. (1976) and Stinson and Glick (1978) for cells derived from chicken thymus, could not be observed in our studies with the SEM. These findings indicate that the SEM is not a useful tool for distinguishing T and B cells in the peripheral blood of the chicken. Surface morphology of lymphocytes in the chicken, e.g., number, size and shape of microvilli, obviously depends on the functionally different cellular status and might represent a variable and dynamic formation of these cells. Furthermore, the SEM-morphology of lymphocytes is strongly influenced by the preparation method used (see Alexander et al., 1976).
References Albini, B., Wick, G.: Immunoglobulin surface determinants on chicken lymphoid cells. Advanc. Exp. Med. Biol. 29, 203-208 (1973) Alexander, E.L., Wetzel, B.: Human lymphocytes: Similarity of B and T cell surface morphology. Science 188, 732-734 (1975) Alexander, E., Sanders, S., Braylan, R.: Purported difference between human T- and B-cell surface morphology is an artefact. Nature 261, 239-241 (1976) Akiyama, Y., Kato, S.: Scanning electron microscopy of lymphoid cell lines and lymphocytes from thymus, bursa and blood of chickens. Biken J. 17, 193-197 (1974) Baur, P.S., Thurman, G.B., Goldstein, A.L.: Reappraisal of lymphocyte classification by means of surface morphology. J. Immunol. 115, 1375-1380 (1975) Boyum, A.: Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest. 21, Suppl. 97, 77-89 (1968) Coudert, F., Richard, J.: The purification oflymphocytes in chicken blood. Poultry Sci. 54, 59-63 (1975) Ewijk, W. van, Brons, N.H.C.: Scanning electron microscopy of B and T cells in peripheral lymphoid organs of the mouse. Adv. Exp. Biol. Med. 66, 171-175 (1976) Dantchev, D., Belpomme, D.: Critical study of the mononuclear leukocyte morphology based on scanning electron microscopy in normal subjects and in patients with lymphoid or monocytoid proliferative disorders. Comparison with the T, B or null cell membrane phenotypes. Biomedicine 26, 202-222 (1977)
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Hoffmann-Fezer, G., L6sch, U., Hoffmann, R., Schmahl, W.: Decreased surface immunoglobulins on lymphoeytes of genuine dysgammaglobulinemicchicken. Z. Immunitaetsforsch. 147, 61-68 (1974) Ivanyi, J., Hudson, L.: Allelic exclusion of M 1 (IgM) allotype on the surface of chicken B ceils. Immunology 35, 941-945 (1979) Ivanyi, J., Fuensalida, E., Lydyard, P.M.: Rapid recovery of antigen-binding receptors on chicken B cells following anti-Ig serum treatment. Eur. J. Immunol. 6, 25-30 (1976) Linthicum, D.S., Sell, S., Wagner, R.M., Trefts, P.: Scanning immunelectron microscopy of mouse B and T lymphocytes. Nature 252, 173-175 (1974) Nazerian, K., Ackerson, A., Hooper, G.: Scanning electron microscopy in the study of chicken T and B cells and cells from Marek's disease tumours. Avian Pathol. 5, 135-145 (1976) Noble, P.B., Cutts, H.: Isolation of individual leukocyte types from peripheral blood. J. Lab. Clin. Med. 72, 533-538 (1968) Polliack, A.: Normal, transformed and leukemic leukocytes. A scanning electron microscopy atlas. Berlin-Heidelberg-New York: Springer Verlag 1977 Polliack, A., Lampen, N., Clarkson, B.D., de Harven, E., Bentwick, Z., Siegal, F.P., Kunkel, H.G.: Identification of human B and T lymphocytes by scanning electron microscopy. J. Exp. Med. 138, 607-624 (1973) Polliack, A., H~immerling, U., Lampen, N., de Harven, E.: Surface morphology of murine B and T lymphocytes: A comparative study by scanning electron microscopy. Eur. J. Ima'nunol. 5, 32-39 (1975) Stinson, R., Glick, B.: Scanning electron microscopy of chicken lymphocytes: A comparative study of thymic, bursal, and splenic lymphocytes. Dev. Comp. Immunol. 2, 311-318 (1978) Accepted October 8, 1979
Cell and Tissue Research 9 by Springer-Verlag 1979
Scanning Electron Microscopy of Peripheral Blood Leukocytes of the Chicken Eberhard Burkhardt Institut fiir Veterinfir-Pathologieder Justus Liebig-Universit~it Giessen, Giessen, Bundesrepublik Deutschland
Summary.Polymorphonuclear leukocytes, e.g., neutrophilic granulocytes, were .enriched from heparinized blood by a Ficoll-step-gradient centrifugation procedure. Scanning electron microscopy (SEM) revealed a surface morphology of narrow ridge-like profiles and small ruffles with occasional microprocesses. Mononuclear leukocytes were isolated by centrifugation over a Ficoll-Metrizoat gradient. The lymphocytes showed varying numbers of microvilli of different length, size and shape. B lymphocytes, characterized by their capability of"sheep red blood cell (SRBC)-rosette formation", displayed a similar surface morphology. Completely smooth lymphocytes, described in the literature as T lymphocytes, could not be detected, although many lymphocytes with few microprocesses were observed. Thus, SEM is not a useful tool for distinguishing between B and T lymphocytes in the peripheral blood of chickens. Monocytes were characterized by prominent membrane-like ruffles, but in some cases they closely resembled granulocytes. An influence of the various separation media on the surface morphology of the isolated cells could not be detected when compared with cells isolated by the buffy-coat method. Key words: Granulocytes - Lymphocytes - Monocytes - Scanning electron microscopy - Chicken.
Efforts to distinguish B from T lymphocytes scanning electron microscopically (SEM) have been controversial. Some authors have described a clear-cut surface morphology of human and murine B and T cells (Polliack et al., 1973, 1975; Linthicum et al., 1974; van Ewijk and Brons, 1976; Polliack, 1977). B cells show a rough surface with many microvilli, while T cells reveal a relatively smooth cell surface. Other authors have been unable to confirm these findings (Alexander and Send offprint requests to: Dr. Eberhard Burkhardt, Institut for Veterin~ir-Pathologieder Justus Liebig-
Universit~it, Frankfurter StraBe 96, D-6300 Giessen, Federal Republic of Germany Acknowledgements: This study was supported by the SFB 47 of the Deutsche Forschungsgerneinschaft, Federal Republic of Germany. The author thanks Mrs. U. Zeller for photographical assistance
0302-766X/79/0204/0147/$01.40
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W e t z e l , 1975; B a u r et al., 1975; A l e x a n d e r et al., 1976; D a n t c h e v a n d B e l p o m m e , 1977). I n the c h i c k e n , A k i y a m a a n d K a t o (1974) a n d N a z e r i a n et al. (1976) f o u n d a r o u g h s u r f a c e o f cells d e r i v e d f r o m the b u r s a o f F a b r i c i u s , w h i l e cells f r o m the thymus showed a smooth surface without microprocesses. In contrast, Stinson and G l i c k (1978) o b s e r v e d the s m o o t h - c e l l t y p e to be the m o s t p r o m i n e n t , r e g a r d l e s s o f the o r i g i n o f the l y m p h o c y t e s . I n the p e r i p h e r a l b l o o d o f the c h i c k e n , A k i y a m a a n d K a t o (1974) o b s e r v e d t h a t a b o u t 50 % o f t h e l y m p h o c y t e s h a v e a r o u g h s u r f a c e morphology with many long microvilli. I n the p r e s e n t p a p e r the S E M - m o r p h o l o g y o f p e r i p h e r a l b l o o d l e u k o c y t e s o f the c h i c k e n , s e p a r a t e d b y d i f f e r e n t m e t h o d s for o b t a i n i n g s u b g r o u p s o f l e u k o c y t e s , is d e s c r i b e d . F u r t h e r m o r e , t h e s u r f a c e m o r p h o l o g y o f l y m p h o c y t e s w a s inv e s t i g a t e d in a n e f f o r t to d i s t i n g u i s h b e t w e e n T a n d B l y m p h o c y t e s .
Materials and Methods Animals and Blood. Heparinized (50 I.U./ml) blood was taken from the wing vein of 6 month-old White Leghorn chickens. Enrichment ofPolymorphonuclear Leukocytes. A Ficoll-gradient-step centrifugation method described by Noble and Cutts (1968) was used. Briefly, 5 ml of heparinized blood was layered over 9 ml of a 35 % Ficoll (MG 400000 ) solution in Hank's buffered salt solution (HBSS) and spun at 1000 g for 30 min. The band of white cells on the top of the gradient was washed once in HBSS and layered over a Ficoll-step gradient ranging from 35 % Ficoll solution in HBSS in the bottom layer to 14.5 % Ficoll solution in HBSS in the top layer. The cells were spun at 50g for 5min followed by 300g for 10min. After centrifugation polymorphonuclear leukocytes were found in the 24% and 18 % Ficoll-gradient steps, from which they were removed by use of a Pasteur pipette. Isolation ofMononuclear Leukocytes. The Ficoll-Metrizoat-gradient centrifugation method (Boyum, 1968; Coudert and Richard, 1975) was used. 5 ml of heparinized blood was layered over 2 ml FicollRonpacon | separation medium and spun at 600 g for 20min in a 12ml glass centrifuge tube. The isolated mononuclear leukocytes were found in a small opaque ring at the interface of the separation medium and plasma, from which they were removed with a Pasteur pipette. Isolation of Total White Blood Cells in a Buffy Coat. 10 ml of heparinized blood were spun at 1500 g for 10 min. The resulting buffy coat containing all white blood cells was removed from the underlying red cell column by means of a Pasteur pipette. Isolation of B Lymphoeytes by Sheep Red Blood Cell (SRBC)-Rosette Formation. For SRBC-rosette formation the method described by Ivanyi et al. (1976) was used. Briefly, 6 days after intravenous immunization with 3 % SRBC suspension in HBSS (1 ml/kg body weight), 30 ml of heparinized blood was taken from a chicken by vein puncture. The mononuclear leukocytes were isolated by FicollMetrizoat-gradient centrifugation. 0.1 ml of a washed 2 % SRBC suspension in HBSS was mixed with an equal volume of the isolated cell suspension (2 • l0 Tcells/ml) in glass centrifuge tubes with round bottoms and kept on ice for 5 min. The tubes were spun at 200 g for 7 min and kept on ice again for 30min. The cell pellets were gently resuspended, pooled and suspended in HBSS to a total volume of 5 ml, layered over 2 ml of FicoU-Ronpacon| and spun at 600 g for 20 min. Then the SRBC rosetteforming cells were found in the red cell column at the bottom of the centrifuge tube, while the nonrosette-forming mononuclear leukocytes were found together with a few SRBC in a small ring at the interface of the separation medium and overlying buffer. Both cell fractions were prepared for SEM. Preparation of Cells for SEM. Cells obtained by the various separation methods were washed twice (200 g, 5 min) in HBSS, resuspended in 1 ml HBSS and injected with a Pasteur pipette into 10 ml of 1.2 % glutaraldehyde in 0.1 M cacodylate buffer (pH = 7.3, osmolality = 300 mO smol) and fixed for 1 h. The
Peripheral Blood Leukocytes (Chicken)
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cells were washed twice in 0.16M cacodylate buffer (pH = 7.3, osmolality = 300mOsmol) and postfixed in 1 ~ OsO4 in 0.1 M cacodylate buffer (pH = 7.3, osmolality = 225 mOsmol) for 1 h. The cells were dehydrated in a graded series of ethanol and in three fractions of absolute ethanol (minimum time of 10 min for each step). The cells were then processed through mixtures of ethanol/iso-amyl acetate 3 : 1, 1 : 1, I : 3 and through two fractions of pure iso-amyl acetate. Three drops of this cell suspension were brought onto small aluminium membrane dishes with a diameter o f about 10 mm and allowed to settle for 2 min. The cells were critical-point dried in CO 2 in a Balzers Union Critical Point Dryer, the aluminium dishes attached to aluminium stubs with a carbon-propanol glue and coated with gold in a Balzers Union Sputtering device. The preparations were examined with a Siemens Autoscan at 20 kV and a tilt angle of 45 ~ Photographs were taken with Agfapan Professional 25 film.
Light Microscopy. Giemsa-stained smears prepared from each cell suspension obtained by the various separation procedures were used as controls.
Fig. l a d . Scanning electron micrographs, a Aggregated granulocytes, x 4200. b Granulocyte, x 10,600. c Granulocyte with small microprocesses (arrows), x 10,500. d Monocyte with broad and prominent ruffles (arrows), x 12,600
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Fig. 2a-i. Lymphocytes showing microvilli of different number, length and shape. Highly villous forms: a x 11,000; b x 9600; e x 9800. Less villous forms: d • 8000; e x 10,600; f x 9700. Relatively smooth forms: g • 10,000; h • 9400; i x 9700
Results A m o n g Ficoll gradient-step and buffy coat-separated cells m a n y spherical cells with a diameter o f 5-6 ~tm were found, which tended to aggregate (Fig. 1 a). Their cell surface showed n a r r o w ridge-like profiles and small ruffles (Fig. l a-c) and occasionally a few microprocesses (Fig. lc, arrows). In Giemsa-stained smears these cells were identified as p o l y m o r p h o n u c l e a r leukocytes; m o s t o f them were heterophilic granulocytes. Some cells with a similar surface m o r p h o l o g y , but m o r e prominent, membrane-like ruffles (Fig. 1 d, arrows), were f o u n d in the buffy coat and in Ficoll-Metrizoat-separated cell suspensions. These cells were identified as monocytes in Giemsa-stained smears. Most o f the Ficoll-Metrizoat-separated cells were identified as lymphocytes in Giemsa-stained smears. They were 3-5 ktm in diameter and had a varying n u m b e r o f microvilli (Fig. 2a-i). Very few cells
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Fig. 3a and b. SRBC rosette-formingcells (B-lymphocytes)displayingdifferentsurface morphology. a x5400; b x5600
displayed rather long "spiky" microvilli (Fig. 2g), while most of them showed relatively short microvilli. Some of these were stud-like (Fig. 2e) or "coral"-shaped (Fig. 2 a), while others were finger-like (Fig. 2 b, c, d). There were large differences in the number of microvilli per cell. Cells with a highly villous (Fig. 2 a-c), a less villous (Fig. 2d-f) and a relatively smooth cell surface (Fig. 2g-i) were encountered. The SRBC rosette-forming cells displayed microvilli of varying numbers and shape and were attached to some spheroechinocytic erythrocytes (Fig. 3a, b). Some rosetteforming cells possessed a coral-like surface (Fig. 3 a), while others showed a highly villous cell surface characterized by finger-like microvilli (Fig. 3b). All SRBC rosette-forming cells were identified light microscopically as lymphocytes; they showed the same SEM-morphology as the lymphocytes not forming SRBC rosettes. Differences in surface morphology of the various subgroups of leukocytes after isolation with the buffy-coat method and with the gradient media were not observed.
Discussion
In the present study, the enriched polymorphonuclear leukocytes of the chicken were characterized by ridge-like profiles and small ruffles. Their surface morphology was similar to that of human neutrophilic granulocytes described by Polliack (1977), while human eosinophils and basophils reveal a different surface structure (Polliack, 1977). From the present studies, it could not be conclusively determined that heterophilic granulocytes differ in their surface morphology from eosinophils and basophils, because we were not able to separate these subgroups of granulocytes. A few cells with a similar structure of the surface, but with broader and more prominent ruffles were observed in Ficoll-Metrizoat gradient and in buffy coat-separated cell suspensions. These cells were characterized in Giemsa-
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E. Burkhardt
stained smears as monocytes. The same SEM-surface morphology was described for monocytes in human blood by Polliack (1977). Sometimes, these cells were confused with polymorphonuclear leukocytes in the SEM. Lymphocytes showed a surface morphology different from that of the abovedescribed cell elements. They are characterized by varying numbers ofmicrovilli of different length and shape. However, a clear-cut surface morphology for two different subgroups of lymphocytes, as described in the chicken, for bursa and thymus cells by Akiyama and Kato (1974) and Nazerian et al. (1976), was not observed. These authors emphasized that bursal cells possess a villous surface. In our studies the cell surface morphology of peripheral blood lymphocytes varied continuously from highly villous, less villous, to relatively smooth forms. However, the percentage of B lymphocytes in chicken peripheral blood ranges from 5-15 ~, as has been indicated by the presence of surface immunoglobulin (Albini and Wick, 1973; Hoffmann-Fezer et al., 1973; Ivanyi and Hudson, 1978). SRBC rosetteforming cells, which were characterized as B lymphocytes by Ivanyi et al. (1976), also varied from highly villous (Fig. 3 b) forms to cells with fewer microvilli. Lymphocytes with a completely smooth cell surface and lacking microvilli, as described by Akiyama and Kato (1974), Nazerian et al. (1976) and Stinson and Glick (1978) for cells derived from chicken thymus, could not be observed in our studies with the SEM. These findings indicate that the SEM is not a useful tool for distinguishing T and B cells in the peripheral blood of the chicken. Surface morphology of lymphocytes in the chicken, e.g., number, size and shape of microvilli, obviously depends on the functionally different cellular status and might represent a variable and dynamic formation of these cells. Furthermore, the SEM-morphology of lymphocytes is strongly influenced by the preparation method used (see Alexander et al., 1976).
References Albini, B., Wick, G.: Immunoglobulin surface determinants on chicken lymphoid cells. Advanc. Exp. Med. Biol. 29, 203-208 (1973) Alexander, E.L., Wetzel, B.: Human lymphocytes: Similarity of B and T cell surface morphology. Science 188, 732-734 (1975) Alexander, E., Sanders, S., Braylan, R.: Purported difference between human T- and B-cell surface morphology is an artefact. Nature 261, 239-241 (1976) Akiyama, Y., Kato, S.: Scanning electron microscopy of lymphoid cell lines and lymphocytes from thymus, bursa and blood of chickens. Biken J. 17, 193-197 (1974) Baur, P.S., Thurman, G.B., Goldstein, A.L.: Reappraisal of lymphocyte classification by means of surface morphology. J. Immunol. 115, 1375-1380 (1975) Boyum, A.: Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest. 21, Suppl. 97, 77-89 (1968) Coudert, F., Richard, J.: The purification oflymphocytes in chicken blood. Poultry Sci. 54, 59-63 (1975) Ewijk, W. van, Brons, N.H.C.: Scanning electron microscopy of B and T cells in peripheral lymphoid organs of the mouse. Adv. Exp. Biol. Med. 66, 171-175 (1976) Dantchev, D., Belpomme, D.: Critical study of the mononuclear leukocyte morphology based on scanning electron microscopy in normal subjects and in patients with lymphoid or monocytoid proliferative disorders. Comparison with the T, B or null cell membrane phenotypes. Biomedicine 26, 202-222 (1977)
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Hoffmann-Fezer, G., L6sch, U., Hoffmann, R., Schmahl, W.: Decreased surface immunoglobulins on lymphoeytes of genuine dysgammaglobulinemicchicken. Z. Immunitaetsforsch. 147, 61-68 (1974) Ivanyi, J., Hudson, L.: Allelic exclusion of M 1 (IgM) allotype on the surface of chicken B ceils. Immunology 35, 941-945 (1979) Ivanyi, J., Fuensalida, E., Lydyard, P.M.: Rapid recovery of antigen-binding receptors on chicken B cells following anti-Ig serum treatment. Eur. J. Immunol. 6, 25-30 (1976) Linthicum, D.S., Sell, S., Wagner, R.M., Trefts, P.: Scanning immunelectron microscopy of mouse B and T lymphocytes. Nature 252, 173-175 (1974) Nazerian, K., Ackerson, A., Hooper, G.: Scanning electron microscopy in the study of chicken T and B cells and cells from Marek's disease tumours. Avian Pathol. 5, 135-145 (1976) Noble, P.B., Cutts, H.: Isolation of individual leukocyte types from peripheral blood. J. Lab. Clin. Med. 72, 533-538 (1968) Polliack, A.: Normal, transformed and leukemic leukocytes. A scanning electron microscopy atlas. Berlin-Heidelberg-New York: Springer Verlag 1977 Polliack, A., Lampen, N., Clarkson, B.D., de Harven, E., Bentwick, Z., Siegal, F.P., Kunkel, H.G.: Identification of human B and T lymphocytes by scanning electron microscopy. J. Exp. Med. 138, 607-624 (1973) Polliack, A., H~immerling, U., Lampen, N., de Harven, E.: Surface morphology of murine B and T lymphocytes: A comparative study by scanning electron microscopy. Eur. J. Ima'nunol. 5, 32-39 (1975) Stinson, R., Glick, B.: Scanning electron microscopy of chicken lymphocytes: A comparative study of thymic, bursal, and splenic lymphocytes. Dev. Comp. Immunol. 2, 311-318 (1978) Accepted October 8, 1979
