- Email: [email protected]
Surface morphology of normal and chronic lymphocytic leukaemia lymphocytes
Euro!. J. Cancer Vol. 12, pp. 199-202.
Pergamon
Press 1976.
Printed
in Great
Britain
Surface Morphology of Normal and Chronic Lymphocytic Leukaemia Lymphocytes* G. COHNEN,t
K. FISCHER,?
TDivision of Haematology,
H. LUDWIG:
and
G. BRITTINGERt
Department of Medicine, and IDepartment of Gynaecology and Obstetrics, University of Essen, Essen, Germany
Abstract-Peripi’leral blood lymphocytes from normals and patients with chronic &mphocytic leukaemia (CLL) were studied by scanning electron microscopy. The surface morphology of normal lymphocytes showed considerable heterogeneity. Most cells had a small or moderate number of stub- or jinger-like projections. In contrast, CLL lymphocytes exhibited a rather homogeneous surface architecture indicating the monoclonal nature of the neopla.stic population. The dominating cell type either had sparse microvilli or was moderately to markedly villous in 9 of the 10 cases studied. Lymphocytes from one patient displayed a smooth, albeit slightly irregular, surface. The abnormality of leukaemic lymphocytes was further indicated by their reduced cell size.
INTRODUCTION
CLL patients in comparison with normal using scanning electron microscopy.
SCANNING electron microscopy has been applied in previous stuidies to investigate the surface morphology of various human blood cells the surface architecture of [l-4]. Recently, normal human lymphocytes has been examined by this method, and differences have been described between thymus-dependent (T) and bone marrow-dependent (B) cells [5]. In chronic lymphocytic leukaemia (CLL), lymphocytes are proliferating and accumulating which exhibit membrane-bound immunoglobulins, a surface marker for B cells [6-91. As seen by transmission electron microscopy, CLL lymphocytes differ from normal cells by a reduced size [lo-121 and by a diminished number of lysosomes [ 131. Furthermore, various membrane abnormalities have been found in the leukaemic cells [14-l 71. In order to define possible alterations of their surface morphology, we studied peripheral blood lymphocytes from
cells
MATERIAL AND METHODS Lymphocytes were separated from heparinized venous blood of 8 normal donors and of 10 patients with CLL (lymphocyte counts ranging from 16,000 to 130,000/4) by centrifugation over a Ficoll-sodium metrizoate solution (D = 1.077) at 400 g for 40 min. Viability as judged by dye exclusion test using erythrosin B was > 95%. Differential counts of stained smears revealed that preparations from normals contained > 95% mononuclear cells with 7080% lymphocytes. Purity of the CLL lymphocyte suspensions was > 95%. At light microno morphological differences were scopy, observed between the cells from the different CLL patients. As determined by spontaneous rosette formation with sheep red blood cells (SRBC), 68-80% of normal lymphocytes and l-12% of CLL lymphocytes were T cells. About 80-90x of the blood lymphocytes from the CLL patients studied have previously been shown to bear B cell markers by immunofluorescent
Accepted 15 October 1975. *This work was supported by Ministerium fiir Wissenschaft und Forsctmng, Nordrhein-Westfalen, Deutsche Forschungsgemeinschaft (LU 118-6), and Alfred und Glare Pott-Stiftung, Essen, Germany.
I99
200
G. Cohnen, K. Fischer, H. Ludwig and G. Brittinger
staining of membrane-bound
immunoglobulins
PI-
electron microscopy, the For scanning lymphocytes were fixed at room temperature in 2% redistilled glutaraldehyde in phosphate buffer (pH 7.4; 320 mosm) and were kept in the fixative for at least 12 hr at 4°C. Then, the cells were layered on polycarbonate membrane filters of 0.8 pm porosity (Nuclepore; Thomas Co., Philadelphia, Pa., U.S.A.) by aspirationfiltration. Premature drying of the cells was always carefully avoided during the subsequent preparatory procedure. After rinsing with phosphate buffer (pH 7.4), the cells were dehydrated in graded concentrations of aethyl alcohol followed by a graded series of amyl acetate/absolute alcohol and finally absolute amyl acetate each for 1 hr at 22°C. After critical point drying in carbon dioxide (Polaron E 3000; Polaron Equipment Ltd., London, England), portions of the filters were attached to specimen holders using double-sided sticky tape. Then, the cells were coated with a thin layer of gold by a sputtering method in a vacuum apparatus (Balzers AG, Liechtenstein) and were examined at an angle of 30” in a Cambridge Stereoscan S 4-l 0 scanning electron microscope (Scientific and Medical Instruments Ltd., Cambridge, England) which was used at an accelerating voltage of 20 kV and with a 200 pm dia illuminating aperture. The resolution of the microscope was in the order of 150 A. Hundreds of cells were scanned on the screen at direct magnifications of from 1000 to 20,000 to obtain an evaluation of cell size and surface morphology of the entire population before recording micrographs which were used for more detailed measurements.
RESULTS Normal peripheral blood lymphocytes showed different kinds of membrane morphology. About 80% of the cells which ranged from 4-O to 6~0 pm (mean 4.8 /*m) dia had a small to moderate number of surface projections (Fig. 1, 2). These were either stub-like (70-200 nm broad) or short microvilli (up to 400 nm long, 100-300 nm broad). Their number varied from cell to cell between 10 and 50 (mean 35) per exposed half of the surface. About 15% of the lymphocytes (mean dia 5.5 pm) were characterized by a more complex surface architecture with 50-150 (mean 85) microvilli per exposed part of the cell (Fig. 2). These projections varied in length from O-5 to 1-O pm and in breadth from 100 to 300 nm. Lympho-
cytes with microvilli covering almost the entire membrane were rarely seen. Single cells were completely devoid of surface digitations. Monocytes were identified by their ruffled membranes and ridge-like profiles. Similar to normal cells, all CLL lymphocytes were spherical with the method used. Cells from 7 patients had a diameter ranging from 3.8 to 5-O pm (mean 4.2 ,nm) and showed a small or moderate number of surface projections (up to 50 microvilli per exposed surface) which was almost constant on the majority of lymphocytes in each of the cases (Fig. 3, 4). The size of the microvilli was similar to that on corresponding normal cells. In 2 patients the lymphocytes had a dia of 4.2-5.2 pm (mean 4.8 pm) and displayed a more villous surface with about 80 processes per exposed part of the cell (Fig. 5). Cells from one patient which ranged from 3.7 to 4.4 pm (mean 4-O pm) dia had a smooth surface with some degree of irregularity (Fig. 6). In each of the patients studied more than 90% of the lymphocytes belonged to the same morphological type. Only single lymphocytes were observed which differed in their surface morphology from most other cells (Fig. 6). The 2 smooth cells shown in Fig. 3 may be interpreted as lymphocytes which are completely devoid of microvilli. However, the possibility cannot be excluded that they represent spherocytes. DISCUSSION Our observations indicate that most normal human blood lymphocytes display a small to moderate number of stub- or finger-like surface projections. Previous studies have shown that T cells possess smaller numbers of microvilli and are generally smoother than the more villous B cells [5]. Similarly, we found about 80% smoother cells among preparations of normal peripheral blood lymphocytes which contained up to 80% SRBC rosette-forming T cells. However, despite this coincidence it is difficult to accurately identify T and B lymphocytes merely on the basis of these morphological criteria, since there exists an overlapping population with intermediate surface architecture [ 181. Furthermore, temperature [ 191, cell cycle [20], and cellular contact and interaction in vitro [21-231 have been demonstrated to influence the surface appearance of the cells. Thus, the possibility has to be considered that the morphological features may reflect different functional states of the lymphocytes rather than specific membrane properties of either T or B cells.
f“i,q.
Spherical normal [ymphocyte showing n moderate uumber qf microvilli (‘2lug. x 10,930j. zyig. 2. 7‘wo normal !ymphocytes with a amnll number of club-llhr /lrqjectiorrs aud one cell with a markedly r*illou\ .suTface (.Zlag. x 5670). l;iCq. 3. S’/)hericcrl CLL lymphocytes .rhowing a small number qf‘micror’illi. Furthermore, two cells with a smooth swface nnd a,, erythrocyte are .FPP~iAZlag. x $050).
I.
Surface Morphology of Lymphocytic Leukaemia Lymphocytes The surface morphology of cells may depend on the preparatory procedures used. Thus, the attachment of live cells to the filter membranes prior to fixation alters their surface structure [24]. In the present study, the lymphocytes have therefore been first fixed in suspension and then layered on the filters. Although the adherence of these prefixed cells is reduced [24], there is no evidence as yet that the resulting cell loss is selective. Moreover, the morphological features of the cells may be influenced by the osmotic pressure of the fixative solution [25] or by some of the methods used to collect and separate the lymphocytes
P81.
It is now well established that the majority of blood lymphocytes from most patients with CLL bear membrane-bound immunoglobulins which are restricted to one heavy and light chain, usually IgM K or IgM 1, suggesting a monoclonal proliferation of the leukaemic cells [6, 8, 91. This concept is further supported by our morphological findings. Thus, in contrast to normal cells;, CLL lymphocytes from the individual patients represent a rather homogeneous population with regard to their surface architecture. T:hose single lymphocytes which differ in their surface morphology from the dominating (leu kaemic) cells most likely belong to the residual population of normal lymphocytes. In most cases the predominant cell type is characterized. either by a moderately or by a markedly villous surface. The presence of microvilli on leukaemic lymphocytes has recently also been described by Polliack et al. [5, 181 and Catovsky et al. [26]. However, despite the uniformity of B cell-like membrane markers the spectrum of the morphological features may vary from patient to patient and striking differe:nces may exist in the surface
architecture of CLL lymphocytes. Thus, as demonstrated in the present study, in single cases with B-type CLL the lymphocytes are completely smooth without obvious prdjections. Since all cells have been prepared under standardized conditions, this morphological diversity appears not to result from variations in the technique, but rather seems to reflect membrane alterations and/or different stages of differentiation of the leukaemic cells. The abnormality of CLL lymphocytes is further indicated by the observation that their diameter is reduced in comparison with the corresponding normal cell type. Previous planimetric measurements have shown that this diminution of cell size is attributable to a decreased cytoplasmic content [lo-l 21. It should be mentioned that the diameters of both normal and CLL lymphocytes at scanning electron microscopy are smaller than those obtained at light or transmission electron microscopy. This finding may be caused by a certain degree of cell shrinkage resulting from the critical point drying procedure [24, 271 or by osmotic effects during fixation [25]. Evidence from our findings suggests that the surface morphology of B cell-like CLL lymphocytes cannot be used as a typical example for that of normal B cells and that scanning electron microscopy does not contribute to the determination of the T or B cell nature of leukaemic cells. Further studies are necessary to relate certain patterns of surface structure to the clinical course of the disease and to define possible variations of the morphological features which may occur during progression of the disease or as a consequence of therapy. Acknowledgements-We Metzger and Mrs. M. assistance.
are grateful to Miss H. Funke for excellent technical
REFERENCES 1. 2. 3.
4. 5.
6.
M. BESSIS,Living Blood Cells and their Ultrastructure. (Edited by M. BESSIS) Springer Verlag, Berlin-Heidelberg-New York (1973). J. A. CLARKE and A. J. SALSBURY,Surface ultramicroscopy of human blood cells. Nature (Lond.) 215, 402 (1967). L. W. MCDONALDand T. L. HAYES, Correlation of scanning electron microscope and light microscope images of individual cells in human blood and blood clots. Exp. mol. Path. 10, 186 (1969). T. W. MICHAELIS,N. R. LARRIMER, E. N. METZ and S. P. BALCERZAK, Surface morphology of human leukocytes. Blood 37, 23 (1971). A. POLLIACK,N. LAMPEN,B. D. CLARK~ON and E. DE HARVEN,Identification of human B and T lymphocytes by scanning electron microscopy. J. ex#. Med. 138, 607 (1973). A. C. AGENBERG,K. J. BLOCHand J. C. LONO,Cell-surface immunoglobulins in chronic lymphocytic leukemia and allied disorders. Amer. J. Med. 55, 184 (1973).
201
202
G. Cohnen, K. Fischer, H. Ludwig and G. Brittinger G. COHNEN,W. AUGENER,A. BUKA and G. BRITTINGER,Rosette-forming lymphocytes in normals and patients with malignant lymphomas. Actu haemat. (Basel) 51, 65 (1974). S. S. FR~LAND,J. B. NATVIGand P. STAVEM,Immunological characterization 8. of lymphocytes in lymphoproliferative diseases. Restriction of classes, subclasses, and Gm allotypes of membrane-bound Ig. &and. J. Immunol. 1, 351 (1972). 9. J. L. PREUD’HOMME and M. SELIGMANN, Surface bound immunoglobulins as a cell marker in human lymphoproliferative diseases. Blood 40, 777 (1972). 10. G. COHNEN,S. D. DOUGLAS,E. K~NIG and G. BRITTINGER, Pokeweed mitogen response of lymphocytes in chronic lymphocytic leukemia: A fine structural study. Blood 42, 591 (1973). 11. R. SHREK, Ultrastructure of blood lymphocytes from chronic lymphocytic and lymphosarcoma cell leukemia. J. nat. Cancer Inst. 48, 51 (1972). T. K. MAUGEL and K. D. DAVIS, The lymphocyte of 12. H. R. SCHUMACHER, chronic lymphatic leukemia. I. Electron microscopy-onset. Cancer (Philad.) 26, 895 (1970). 13. S. D. DOUGLAS,G. COHNEN,E. K&no and G. BRITTINGER,Lymphocyte lysosomes and lysosomal enzymes in chronic lymphocytic leukemia. Blood 41, 511 (1973). 14. P. J. L. HOLT, S. G. PAL, D. CATOVSKYand S. M. LEWIS,Surface structure of normal and leukaemic lymphocytes. I. Effects of mitogens. Clin. exp. Immunol. 10, 555 (1972). 15. C. HUBER,G. MICHLMAYR,H. BRAUNSTEINER and H. HUBER,Redistribution of immunoglobulin determinants on human lymphocytes in lymphoproliferative disorders. Europ. J. Cancer 10, 517 (1974). 16. S. KORNFELD, Decreased phytohemagglutinin receptor sites in chronic lymphocytic leukemia. Biochim. biophys. Acta 192, 542 (1969). 17. H.-D. MENNE and H.-D. FLAD, Membrane dynamics of HL-A-anti-HL-Acomplexes of human normal and leukaemic lymphocytes. Clin. ext. Immutwl. 14, 57 (1973). 18. A. POLLIACKand E. DE HARVEN, Surface features of normal and leukemic lymphocytes as seen by scanning electron microscopy. Clin. Immunol. Immunopath. 3, 412 (1975). 19. P. S. LIN, D. F. H. WALLACHand S. TSAI, Temperature-induced variations in the surface topology of cultured lymphocytes are revealed by scanning electron microscopy. Proc. nut. Acad. Sci. (Wash.) 70, 2492 (1973). 20. K. PORTER, D. PRESCOTTand J. FRYE, Changes in surface morphology of Chinese hamster ovary cells during the cell cycle. J. Cell Biol. 57, 815 (1973). 21. M. M. KAY, B. BELOHRADSKY, K. YEE, J. VOGEL, D. BUTCHER,J. WYBRAN and H. H. FUDENBERG, Cellular interactions: Scanning electron microscopy of human thymus-derived rosette-forming lymphocytes. Clin. Immunol. Immunopath. 2, 301 (1974). 22. P. S. LIN, A. G. COOPERand H. H. WORTIS, Scanning electron microscopy of human T-cell and B-cell rosettes. New Engl. J. Med. 289, 548 (1973). 23. A. POLIXACK,S. M. Fu, S. D. DOUGLAS,Z. BENTWICH,N. LAMPENand E. DE HARVEN,Scanning electron microscopy of human lymphocyte-sheep erythrocyte rosettes. J. exp. Med. 140, 146 (1974). 24. B. WETZEL, G. B. CANNON,E. L. ALEXANDER,B. W. ERICKSON JR. and E. W. WESTBROOK, A critical approach to the scanning electron microscopy of cells in suspension. In Scanning Electron Microscopy (Part III). Proceedings of the Workshop on Advances in Biomedical Applications of the SEM, p. 581. IITRI, Chicago (1974). 25. P. B. BELL,JR., U. BRUNK,P. COLLINS,N. FORSBYand B.-A. FREDRIKSSON, SEM of cells in culture: Osmotic effects during fixation. In Scanning Electron Microscopy (Part I). Proceedings of the Eighth Annual Scanning Electron Microscope Symposium, p. 380. IITRI, Chicago (1975). 26. D. CATOVSKY,B. FRISCHand S. VAN NOORDEN,B, T and “null” cell leukaemias. Electron cytochemistry and surface morphology. Blood Cells 1, 115 (1975). 27. A. BOYDE,R. A. WEISSand P. VESELY,Scanning electron microscopy of cells in culture. Exp. Cell Res. 71, 313 (1972). 7.
Pergamon
Press 1976.
Printed
in Great
Britain
Surface Morphology of Normal and Chronic Lymphocytic Leukaemia Lymphocytes* G. COHNEN,t
K. FISCHER,?
TDivision of Haematology,
H. LUDWIG:
and
G. BRITTINGERt
Department of Medicine, and IDepartment of Gynaecology and Obstetrics, University of Essen, Essen, Germany
Abstract-Peripi’leral blood lymphocytes from normals and patients with chronic &mphocytic leukaemia (CLL) were studied by scanning electron microscopy. The surface morphology of normal lymphocytes showed considerable heterogeneity. Most cells had a small or moderate number of stub- or jinger-like projections. In contrast, CLL lymphocytes exhibited a rather homogeneous surface architecture indicating the monoclonal nature of the neopla.stic population. The dominating cell type either had sparse microvilli or was moderately to markedly villous in 9 of the 10 cases studied. Lymphocytes from one patient displayed a smooth, albeit slightly irregular, surface. The abnormality of leukaemic lymphocytes was further indicated by their reduced cell size.
INTRODUCTION
CLL patients in comparison with normal using scanning electron microscopy.
SCANNING electron microscopy has been applied in previous stuidies to investigate the surface morphology of various human blood cells the surface architecture of [l-4]. Recently, normal human lymphocytes has been examined by this method, and differences have been described between thymus-dependent (T) and bone marrow-dependent (B) cells [5]. In chronic lymphocytic leukaemia (CLL), lymphocytes are proliferating and accumulating which exhibit membrane-bound immunoglobulins, a surface marker for B cells [6-91. As seen by transmission electron microscopy, CLL lymphocytes differ from normal cells by a reduced size [lo-121 and by a diminished number of lysosomes [ 131. Furthermore, various membrane abnormalities have been found in the leukaemic cells [14-l 71. In order to define possible alterations of their surface morphology, we studied peripheral blood lymphocytes from
cells
MATERIAL AND METHODS Lymphocytes were separated from heparinized venous blood of 8 normal donors and of 10 patients with CLL (lymphocyte counts ranging from 16,000 to 130,000/4) by centrifugation over a Ficoll-sodium metrizoate solution (D = 1.077) at 400 g for 40 min. Viability as judged by dye exclusion test using erythrosin B was > 95%. Differential counts of stained smears revealed that preparations from normals contained > 95% mononuclear cells with 7080% lymphocytes. Purity of the CLL lymphocyte suspensions was > 95%. At light microno morphological differences were scopy, observed between the cells from the different CLL patients. As determined by spontaneous rosette formation with sheep red blood cells (SRBC), 68-80% of normal lymphocytes and l-12% of CLL lymphocytes were T cells. About 80-90x of the blood lymphocytes from the CLL patients studied have previously been shown to bear B cell markers by immunofluorescent
Accepted 15 October 1975. *This work was supported by Ministerium fiir Wissenschaft und Forsctmng, Nordrhein-Westfalen, Deutsche Forschungsgemeinschaft (LU 118-6), and Alfred und Glare Pott-Stiftung, Essen, Germany.
I99
200
G. Cohnen, K. Fischer, H. Ludwig and G. Brittinger
staining of membrane-bound
immunoglobulins
PI-
electron microscopy, the For scanning lymphocytes were fixed at room temperature in 2% redistilled glutaraldehyde in phosphate buffer (pH 7.4; 320 mosm) and were kept in the fixative for at least 12 hr at 4°C. Then, the cells were layered on polycarbonate membrane filters of 0.8 pm porosity (Nuclepore; Thomas Co., Philadelphia, Pa., U.S.A.) by aspirationfiltration. Premature drying of the cells was always carefully avoided during the subsequent preparatory procedure. After rinsing with phosphate buffer (pH 7.4), the cells were dehydrated in graded concentrations of aethyl alcohol followed by a graded series of amyl acetate/absolute alcohol and finally absolute amyl acetate each for 1 hr at 22°C. After critical point drying in carbon dioxide (Polaron E 3000; Polaron Equipment Ltd., London, England), portions of the filters were attached to specimen holders using double-sided sticky tape. Then, the cells were coated with a thin layer of gold by a sputtering method in a vacuum apparatus (Balzers AG, Liechtenstein) and were examined at an angle of 30” in a Cambridge Stereoscan S 4-l 0 scanning electron microscope (Scientific and Medical Instruments Ltd., Cambridge, England) which was used at an accelerating voltage of 20 kV and with a 200 pm dia illuminating aperture. The resolution of the microscope was in the order of 150 A. Hundreds of cells were scanned on the screen at direct magnifications of from 1000 to 20,000 to obtain an evaluation of cell size and surface morphology of the entire population before recording micrographs which were used for more detailed measurements.
RESULTS Normal peripheral blood lymphocytes showed different kinds of membrane morphology. About 80% of the cells which ranged from 4-O to 6~0 pm (mean 4.8 /*m) dia had a small to moderate number of surface projections (Fig. 1, 2). These were either stub-like (70-200 nm broad) or short microvilli (up to 400 nm long, 100-300 nm broad). Their number varied from cell to cell between 10 and 50 (mean 35) per exposed half of the surface. About 15% of the lymphocytes (mean dia 5.5 pm) were characterized by a more complex surface architecture with 50-150 (mean 85) microvilli per exposed part of the cell (Fig. 2). These projections varied in length from O-5 to 1-O pm and in breadth from 100 to 300 nm. Lympho-
cytes with microvilli covering almost the entire membrane were rarely seen. Single cells were completely devoid of surface digitations. Monocytes were identified by their ruffled membranes and ridge-like profiles. Similar to normal cells, all CLL lymphocytes were spherical with the method used. Cells from 7 patients had a diameter ranging from 3.8 to 5-O pm (mean 4.2 ,nm) and showed a small or moderate number of surface projections (up to 50 microvilli per exposed surface) which was almost constant on the majority of lymphocytes in each of the cases (Fig. 3, 4). The size of the microvilli was similar to that on corresponding normal cells. In 2 patients the lymphocytes had a dia of 4.2-5.2 pm (mean 4.8 pm) and displayed a more villous surface with about 80 processes per exposed part of the cell (Fig. 5). Cells from one patient which ranged from 3.7 to 4.4 pm (mean 4-O pm) dia had a smooth surface with some degree of irregularity (Fig. 6). In each of the patients studied more than 90% of the lymphocytes belonged to the same morphological type. Only single lymphocytes were observed which differed in their surface morphology from most other cells (Fig. 6). The 2 smooth cells shown in Fig. 3 may be interpreted as lymphocytes which are completely devoid of microvilli. However, the possibility cannot be excluded that they represent spherocytes. DISCUSSION Our observations indicate that most normal human blood lymphocytes display a small to moderate number of stub- or finger-like surface projections. Previous studies have shown that T cells possess smaller numbers of microvilli and are generally smoother than the more villous B cells [5]. Similarly, we found about 80% smoother cells among preparations of normal peripheral blood lymphocytes which contained up to 80% SRBC rosette-forming T cells. However, despite this coincidence it is difficult to accurately identify T and B lymphocytes merely on the basis of these morphological criteria, since there exists an overlapping population with intermediate surface architecture [ 181. Furthermore, temperature [ 191, cell cycle [20], and cellular contact and interaction in vitro [21-231 have been demonstrated to influence the surface appearance of the cells. Thus, the possibility has to be considered that the morphological features may reflect different functional states of the lymphocytes rather than specific membrane properties of either T or B cells.
f“i,q.
Spherical normal [ymphocyte showing n moderate uumber qf microvilli (‘2lug. x 10,930j. zyig. 2. 7‘wo normal !ymphocytes with a amnll number of club-llhr /lrqjectiorrs aud one cell with a markedly r*illou\ .suTface (.Zlag. x 5670). l;iCq. 3. S’/)hericcrl CLL lymphocytes .rhowing a small number qf‘micror’illi. Furthermore, two cells with a smooth swface nnd a,, erythrocyte are .FPP~iAZlag. x $050).
I.
Surface Morphology of Lymphocytic Leukaemia Lymphocytes The surface morphology of cells may depend on the preparatory procedures used. Thus, the attachment of live cells to the filter membranes prior to fixation alters their surface structure [24]. In the present study, the lymphocytes have therefore been first fixed in suspension and then layered on the filters. Although the adherence of these prefixed cells is reduced [24], there is no evidence as yet that the resulting cell loss is selective. Moreover, the morphological features of the cells may be influenced by the osmotic pressure of the fixative solution [25] or by some of the methods used to collect and separate the lymphocytes
P81.
It is now well established that the majority of blood lymphocytes from most patients with CLL bear membrane-bound immunoglobulins which are restricted to one heavy and light chain, usually IgM K or IgM 1, suggesting a monoclonal proliferation of the leukaemic cells [6, 8, 91. This concept is further supported by our morphological findings. Thus, in contrast to normal cells;, CLL lymphocytes from the individual patients represent a rather homogeneous population with regard to their surface architecture. T:hose single lymphocytes which differ in their surface morphology from the dominating (leu kaemic) cells most likely belong to the residual population of normal lymphocytes. In most cases the predominant cell type is characterized. either by a moderately or by a markedly villous surface. The presence of microvilli on leukaemic lymphocytes has recently also been described by Polliack et al. [5, 181 and Catovsky et al. [26]. However, despite the uniformity of B cell-like membrane markers the spectrum of the morphological features may vary from patient to patient and striking differe:nces may exist in the surface
architecture of CLL lymphocytes. Thus, as demonstrated in the present study, in single cases with B-type CLL the lymphocytes are completely smooth without obvious prdjections. Since all cells have been prepared under standardized conditions, this morphological diversity appears not to result from variations in the technique, but rather seems to reflect membrane alterations and/or different stages of differentiation of the leukaemic cells. The abnormality of CLL lymphocytes is further indicated by the observation that their diameter is reduced in comparison with the corresponding normal cell type. Previous planimetric measurements have shown that this diminution of cell size is attributable to a decreased cytoplasmic content [lo-l 21. It should be mentioned that the diameters of both normal and CLL lymphocytes at scanning electron microscopy are smaller than those obtained at light or transmission electron microscopy. This finding may be caused by a certain degree of cell shrinkage resulting from the critical point drying procedure [24, 271 or by osmotic effects during fixation [25]. Evidence from our findings suggests that the surface morphology of B cell-like CLL lymphocytes cannot be used as a typical example for that of normal B cells and that scanning electron microscopy does not contribute to the determination of the T or B cell nature of leukaemic cells. Further studies are necessary to relate certain patterns of surface structure to the clinical course of the disease and to define possible variations of the morphological features which may occur during progression of the disease or as a consequence of therapy. Acknowledgements-We Metzger and Mrs. M. assistance.
are grateful to Miss H. Funke for excellent technical
REFERENCES 1. 2. 3.
4. 5.
6.
M. BESSIS,Living Blood Cells and their Ultrastructure. (Edited by M. BESSIS) Springer Verlag, Berlin-Heidelberg-New York (1973). J. A. CLARKE and A. J. SALSBURY,Surface ultramicroscopy of human blood cells. Nature (Lond.) 215, 402 (1967). L. W. MCDONALDand T. L. HAYES, Correlation of scanning electron microscope and light microscope images of individual cells in human blood and blood clots. Exp. mol. Path. 10, 186 (1969). T. W. MICHAELIS,N. R. LARRIMER, E. N. METZ and S. P. BALCERZAK, Surface morphology of human leukocytes. Blood 37, 23 (1971). A. POLLIACK,N. LAMPEN,B. D. CLARK~ON and E. DE HARVEN,Identification of human B and T lymphocytes by scanning electron microscopy. J. ex#. Med. 138, 607 (1973). A. C. AGENBERG,K. J. BLOCHand J. C. LONO,Cell-surface immunoglobulins in chronic lymphocytic leukemia and allied disorders. Amer. J. Med. 55, 184 (1973).
201
202
G. Cohnen, K. Fischer, H. Ludwig and G. Brittinger G. COHNEN,W. AUGENER,A. BUKA and G. BRITTINGER,Rosette-forming lymphocytes in normals and patients with malignant lymphomas. Actu haemat. (Basel) 51, 65 (1974). S. S. FR~LAND,J. B. NATVIGand P. STAVEM,Immunological characterization 8. of lymphocytes in lymphoproliferative diseases. Restriction of classes, subclasses, and Gm allotypes of membrane-bound Ig. &and. J. Immunol. 1, 351 (1972). 9. J. L. PREUD’HOMME and M. SELIGMANN, Surface bound immunoglobulins as a cell marker in human lymphoproliferative diseases. Blood 40, 777 (1972). 10. G. COHNEN,S. D. DOUGLAS,E. K~NIG and G. BRITTINGER, Pokeweed mitogen response of lymphocytes in chronic lymphocytic leukemia: A fine structural study. Blood 42, 591 (1973). 11. R. SHREK, Ultrastructure of blood lymphocytes from chronic lymphocytic and lymphosarcoma cell leukemia. J. nat. Cancer Inst. 48, 51 (1972). T. K. MAUGEL and K. D. DAVIS, The lymphocyte of 12. H. R. SCHUMACHER, chronic lymphatic leukemia. I. Electron microscopy-onset. Cancer (Philad.) 26, 895 (1970). 13. S. D. DOUGLAS,G. COHNEN,E. K&no and G. BRITTINGER,Lymphocyte lysosomes and lysosomal enzymes in chronic lymphocytic leukemia. Blood 41, 511 (1973). 14. P. J. L. HOLT, S. G. PAL, D. CATOVSKYand S. M. LEWIS,Surface structure of normal and leukaemic lymphocytes. I. Effects of mitogens. Clin. exp. Immunol. 10, 555 (1972). 15. C. HUBER,G. MICHLMAYR,H. BRAUNSTEINER and H. HUBER,Redistribution of immunoglobulin determinants on human lymphocytes in lymphoproliferative disorders. Europ. J. Cancer 10, 517 (1974). 16. S. KORNFELD, Decreased phytohemagglutinin receptor sites in chronic lymphocytic leukemia. Biochim. biophys. Acta 192, 542 (1969). 17. H.-D. MENNE and H.-D. FLAD, Membrane dynamics of HL-A-anti-HL-Acomplexes of human normal and leukaemic lymphocytes. Clin. ext. Immutwl. 14, 57 (1973). 18. A. POLLIACKand E. DE HARVEN, Surface features of normal and leukemic lymphocytes as seen by scanning electron microscopy. Clin. Immunol. Immunopath. 3, 412 (1975). 19. P. S. LIN, D. F. H. WALLACHand S. TSAI, Temperature-induced variations in the surface topology of cultured lymphocytes are revealed by scanning electron microscopy. Proc. nut. Acad. Sci. (Wash.) 70, 2492 (1973). 20. K. PORTER, D. PRESCOTTand J. FRYE, Changes in surface morphology of Chinese hamster ovary cells during the cell cycle. J. Cell Biol. 57, 815 (1973). 21. M. M. KAY, B. BELOHRADSKY, K. YEE, J. VOGEL, D. BUTCHER,J. WYBRAN and H. H. FUDENBERG, Cellular interactions: Scanning electron microscopy of human thymus-derived rosette-forming lymphocytes. Clin. Immunol. Immunopath. 2, 301 (1974). 22. P. S. LIN, A. G. COOPERand H. H. WORTIS, Scanning electron microscopy of human T-cell and B-cell rosettes. New Engl. J. Med. 289, 548 (1973). 23. A. POLIXACK,S. M. Fu, S. D. DOUGLAS,Z. BENTWICH,N. LAMPENand E. DE HARVEN,Scanning electron microscopy of human lymphocyte-sheep erythrocyte rosettes. J. exp. Med. 140, 146 (1974). 24. B. WETZEL, G. B. CANNON,E. L. ALEXANDER,B. W. ERICKSON JR. and E. W. WESTBROOK, A critical approach to the scanning electron microscopy of cells in suspension. In Scanning Electron Microscopy (Part III). Proceedings of the Workshop on Advances in Biomedical Applications of the SEM, p. 581. IITRI, Chicago (1974). 25. P. B. BELL,JR., U. BRUNK,P. COLLINS,N. FORSBYand B.-A. FREDRIKSSON, SEM of cells in culture: Osmotic effects during fixation. In Scanning Electron Microscopy (Part I). Proceedings of the Eighth Annual Scanning Electron Microscope Symposium, p. 380. IITRI, Chicago (1975). 26. D. CATOVSKY,B. FRISCHand S. VAN NOORDEN,B, T and “null” cell leukaemias. Electron cytochemistry and surface morphology. Blood Cells 1, 115 (1975). 27. A. BOYDE,R. A. WEISSand P. VESELY,Scanning electron microscopy of cells in culture. Exp. Cell Res. 71, 313 (1972). 7.