Aaron Polliack

Normal, Transformed and Leukemic Leukocytes A Scanning Electron Microscopy Atlas

With 236 Figures

S pringer -Verlag Berlin Heidelberg New York 1977

AARON POLLIACK, M.D. Associate Professor of Medicine Department of Hematology Hadassah University Hospital and Hebrew University Hadassah Medical School Jerusalem, Israel

ISBN-13: 978-3-642-66727-5 e-ISBN-13: 978-3-642-66725-1 DOl: 10.1007/978-3-642-66725-1

Library of Congress Cataloging in Publication Data. Polliack, Aaron. 1939- . Normal, transformed and leukemic leucocytes. Bibliography: p. . Includes index. 1. Leukemia­Atlases. 2. Leukocytes-Atlases. 3. Cell transformation-Atlases. 4. Cancer cells-Atlases. 5. Scanning electron microscope-Atlases. I. Title. RC643.P63 616.1'55'00222 77-14223

This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically those of translation, reprinting, re-use of illustrations, broadcasting, reproduction by photocopying machine or similar means, and storage in data banks. Under § 54 of the German Copyright Law, where copies are made for other than private use, a fee is payable to the publisher, the amount of the fee to be determined by agreement wIth the publisher.

© by Springer-Verlag Berlin Heidelberg 1977. Softcover reprint of the hardcover I st edition 1977

The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use.

This book is gratefully dedicated to my wife LILY for her assistance and constant encouragement

Preface

The scanning electron microscope (SEM) has been used with increasing frequency in recent years to study the surface mor­phology of normal, transformed and malignant leukocytes. Since the original reports on critical point-dried lymphocytes published in 1973, results of other studies using improved methods have been reported giving rise to some controversy in this field and this is discussed in the text of the atlas. Advances in preparatory techniques recorded during the past 3 years have also contributed much to a better understanding of cell surface phenomena as seen under the SEM. The text of the atlas traces the developments in this field chronologically, summarizes the available literature and presents the current situation in the light of the most recent studies in this field. The photographs were selected to illustrate the spectrum of surface morphology of the different cell types obtained from normal individuals and patients with disease states. Hopefully, the atlas will serve as a guide for future studies and as an illustration of what SEM has to offer in providing details of surface architecture.

In an advancing field such as this in which new methods of cell preparation coupled with vast movements in the resolu­tion of the microscope will no doubt produce better results, atlases run the risk of becoming outdated quite quickly. Never­theless an overall review needs to be presented at times, to provide an overview of the current status of knowledge and of advances in the field, particularly for those who are not intimately involved in its development. It is hoped that this atlas fulfills some of these needs and illustrates the exciting progress which has taken place in recent years.

Jerusalem, Israel September 1977

AARON POLLIACK

Acknowledgments

Some of the studies reviewed and illustrated in this atlas were performed during 1972-1974 while the author was working at the Memorial Sloan-Kettering Cancer Center, New York under the guidance of Etienne de Harven, M.D. and in close collaboration with Nina Lampen, B.A. Some of the photo­graphs appearing in this album, taken in that laboratory by Nina Lampen and myself, have been published in earlier re­ports by us as a group and have been reproduced with the kind permission of the various journals concerned. The author is grateul to Etienne de Harven and Nina Lampen for their invaluable assistance during that period. I am also grateful to Drs. B.D. Clarkson, R.A. Good and H.G. Kunkel for their constant encouragement, advice and stimulation at the time of these studies. The skillful photographic assistance of Juan Marchese during that period is also acknowledged.

Since 1974 these studies have been continued by myself independently at the Department of Hematology, Hadassah University Hospital and Hebrew University-Hadassah Medical School, Jerusalem, Israel. The author is most grateful for the assistance given him during this period by Professor G.Izak, M.D. whose constant encouragement has been a source of stimulation for me. The devoted technical assistance of Miriam Froimovici is also acknowledged. The author is grateful to Mr. Moshe Rosenberg of the Medical Photography Depart­ment of the Hebrew University-Hadassah Medical School for preparation of many of the photographs appearing in this atlas. The author is also grateful to Shirley Ben Yosef for devoted secretarial assistance.

The following journals provided permission to reproduce published figures: The American Journal of Medicine, Biomedi­cine, Blut, British Journal of Haematology, Clinical Immunology and Immunopathology, Israel Journal of Medical Sciences, Jour­nal of Experimental Medicine, Laboratory Investigation, Scan­dinavian Journal of Haematology. Dr. Om Johari, IIT Research Institute, Chicago, provided permission for the reproduction of figures previously published in the proceedings of the SEM symposia of 1975 and 1976.

Contents

A. Introduction and Methods (Figs. 1 - 8)

B. Review and General Details of the Techniques Employed to Prepare Leukocytes . . . . . . 5

C. Normal Leukocytes . . . . . . . . . . . . 8 l. Monocytes and Granulocytes (Figs. 9 - 61) 8

2. Lymphocytes and Thymic Cells: Review of the Factors Affecting the Surface Morphology of Lymphocytes (Figs. 62 - 91) . . . . . . . 32

D. Labeling Studies on Lymphocytes (Figs. 92 -104) 53

E. Other Factors Influencing the Surface Morphology of Lymphocytes and Mammalian Cells . . . . .. 63

F . Mitogen-Transformed Lymphocytes (Figs. 105 -124). 64

G. Cultured Cell Lines (Figs. 125 - 158) . . . . . .. 76

H. Surface Features of Leukemic Cells (Figs. 159 - 236) 97

References . 133

Subject Index 139

A. Introduction and Methods

In recent years the scanning electron microscope (SEM) has been used to study the surface morphology of a variety of mammalian cells. This microscope has a great depth of focus, provides a three­dimensional view of cell surface topography and enables examination of a large number of cells. Thus, SEM provides an overall impression of the surface features of a given population of cells and at the same time details of individual cell surfaces can be obtained. An added advantage of this mode of microscopy is that cells can be prepared relatively quickly for examination in the SEM and results can be ob­tained within a day if necessary. Much, however, depends on the method of cell preparation and in particular, the modes of fixation and drying employed in the processing of cells for SEM. Earlier studies on leukocytes by a number of workers [9, 15, 16, 34, 54] employing air-drying techniques, did not provide optimal details of surface mor­phology. Air-drying causes cell distortion and shrinkage with collapse of surface microprojections [10, 82], making it difficult to record details of the surface morphology of the different cell types and to attempt to characterize them on the basis of their structure. Nevertheless, the results of some of the above studies [9, 15, 16, 34, 54] suggested that lymphocytes had a different surface morphology than monocytes and that mitogen-transformed lymphocytes differed from normallympho­cytes [28, 34]. However, details of fine surface structure were lacking.

In 1972, Porter [82] and Boyde [10] and co-workers showed that when cells were processed using freeze-drying or critical point-drying, results were vastly improved. There was better preservation of the surface structure which enabled one to characterize and recognize different types of surface microprojections such as filipodia, microvilli, blebs and ruffled membranes [82]. The critical point-drying technique described by Anderson in 1951 [5] and later by Cohen et al. in 1968 [17] allows cells to dry without the disturbing effect of surface tension and the results are almost optimal. However, a degree of cell shrinkage still occurs, and cytoplasmic cracks and tears are still seen with this technique, particularly in elongated fragile cells attached to and spread­ing onto various substrates. When different leukocytes were prepared by air- and critical point-drying methods, respectively, and then com­pared under the SEM, the superiority of the latter technique was clearly evident [64] (Figs. 1-8) and it was possible, in many cases, to characterize different leukocyte types more accurately on the basis of their surface structure.

2 Critical Point-Drying vs. Air-Drying

Figs. 1--4. Critical point-dried cultured lymphoblastoid cells (right) compared with the same cells which were air-dried (left) showing preservation of surface microprojections on critical point-dried cells ( x 4500- 7600)

4

Critical Point-Drying vs. Air-Drying 3

iiiiii~::=:====- 5

Figs. 5 and 6. High-power photograph of critical point-dried monocyte with convoluted surface and ruffles (above) compared with the same cell type air-dried ( below). Note the shrinkage of the air-dried cell with loss of fine detail (x 16,300)

4 Critical Point-Drying vs. Air-Drying

Figs. 7 and 8. High-power photographs of cultured Iymphoblastoid cells, comparing critical point- and air-drying techniques. Note the lack of surface detail on the air-dried cells (above) and the well-preserved surface architecture on the critical point-dried cell· (below ) ( x 16,000)

B. Review and General Details of the Techniques Employed to Prepare Leukocytes

Leukocytes were generally prepared from buffy coats obtained from normal heparinized venous blood by centrifugation for 5-8 min at 800-1000 rpm. Leukocyte fractions were obtained using the Ficoll­Hypaque density gradient centrifugation technique [11], which sepa­rates erythrocytes and granulocytes from mononuclear cells (lympho­cytes and monocytes). This technique allows one to harvest granulo­cytes and mononuclear cells separately. Mononuclear cell suspensions contain mainly small lymphocytes with varying numbers of monocytes, while the percentage of contaminating polymorphonuclear leukocytes varies ranging from 0-5%. Cultured cells were concentrated, like circu­lating leukocytes, by gentle centrifugation at 800-1000 rpm for 5-8 min. This was followed by resuspension of the cell pellet in the original culture medium or directly in glutaraldehyde fixative or by fixation after sedimentation within the culture flask in situ.

In earlier studies (64-68) the concentrated cells were resuspended in Hanks' balanced salt solution, phosphate buffer (pH 7.3, 310-320 mosmol) or culture medium without fetal calf serum. After a brief centrifugation, cells in suspension were collected onto porous mem­branes or other substrates, in monolayer-like distribution using gentle aspiration. The specimens were then fixed, generally within 2 min, in 1 % phosphate-buffered glutaraldehyde (pH 7.3, 310-320 mosmol). The filters or coverslips were fixed initially at room temperature for up to 1 h, followed by at least 12 h fixation at 40 C in 1 % glutaralde­hyde. Other investigators [43-46, 40, 86] collected cells onto glass or cell monolayers without aspiration and filtration, using similar fixations.

After fixation, cells were briefly rinsed in phosphate buffer followed by post-fixation for an hour in 1 % osmium tetroxide. The latter post­fixation is not necessarily required as we have noted no differences in samples prepared with or without the use of post-fixation in osmium. Dehydration was continued in a graded series of ethanol followed by a graded series of amyl acetate or Freon 113/absolute ethanol, meticulously avoiding premature drying during all these stages. Dehy­dration was followed by critical point-drying using liquid carbon diox­ide [5] or Freon 13 [17] or freeze-drying. Portions of the substrate were then stuck onto aluminium stubs using double-sided sticky tape or silver paint, and then coated with a thin layer (approximately 200 A) of carbon and gold, gold or gold palladium using a vaccum

6 Review and General Details

coater or a sputtering coating device. Samples should be stored under vacuum until they are examined in the SEM.

Recently, it has been shown [2, 3, 6, 37, 57) that variations in the mode and type of fixation and cell collection play an important role in determining the surface structure of a given cell population and result in variations in the number of microprojections evident on the cell surface. Barber and Burkholder [6] compared several fixa­tives in the preparation of cells for SEM and found differences in the number of microprojections depending on the type and osmolality of the fixative and buffer used. Wetzel and co-workers [101] demon­strated that the time interval between collection of cells onto the substrate and their fixation was also of great importance in this re­spect. Alexander and Wetzel [2] and Newell et al. [57] have shown that when circulating peripheral blood lymphocytes are fixed in suspen­sion prior to collection onto substrates, all cells have microvilli, and differ, in this respect, from unfixed lymphocytes collected onto the same substrates in suspension. It should be mentioned, however, that fixation in suspension may sometimes result in cell clumping on the substrate [57, 76] and in these cases it is necessary to coat cells with a thicker layer of carbon and gold in order to avoid charging effects in the SEM.

Newell et al. [57] recommend collecting cells in an adapted BEEM embedding capsule fitted with a Millipore filter, and sedimenting them onto the membrane during critical point-drying in order to avoid loss of microvilli; In addition, Thornthwaite et al. [95] have described a method in which up to 12 different cell samples can be collected onto glass slides utilizing centrifugal cytology and subsequent fixation with glutaraldehyde.

In 1974, Mazia et al. [52, 53] described a new technique for collect­ing cells onto substrates using polylysine-coated substrates. This tech­nique, modified by Sanders et al. [90], apparently diminishes markedly the cell loss from the substrate during the process of fixation, dehydra­tion and critical point-drying. Using this high yield method, cells prefixed in suspension may be collected onto substrates with minimal cell loss. Recently Alexander et al. [3] have shown that the aspiration filtration technique first described by de Harven et al. [21] and used in our earlier studies [64-72] may, under certain conditions, result in the loss of lymphocyte microvilli if cells have not been fixed in cell suspension prior to collection onto filters. From their studies it appears that cells fixed in suspension do not alter with aspiration and filtration.

In the light of these results it seems best to fix cells in suspension prior to collecting them onto substrates and not to use aspiration at all. In view of the results obtained by Barber and Burkholder [6] a careful study on fixatives and fixation procedures used for SEM needs to

Review and General Details 7

be performed in order to compare different fixatives, to assess how variations in their concentration and osmolality influence the surface architecture of leukocytes and to determine what the optimal duration of fixation for the preparation of leukocytes is. Studies on how the mode of action of different fixatives and the effect of cross-linkage of proteins during fixation, may influence the surface architecture and redistribution of leukocyte microvilli, still need to be performed.

c. Normal Leukocytes

1. Monocytes and Granulocytes

Under the SEM, it is frequently possible to distinguish monocytes from lymphocytes. Monocytes(Figs. 9-30) are larger, more irregular cells with prominent ruffled membranes and ridge-like profiles and microvilli are not a prominent feature (Figs. 9-19). These cells can phagocytose latex beads or erythrocytes when placed in contact with them, and this process can be well visualized under the SEM (Fig. 20). Further­more, the monocytes in monocyte-EA rosettes, when studied by SEM, show similar surface features (Figs. 21, 22) and frequently phagocytose the attached erythrocytes. Like macrophages, monocytes attach to glass and spread, becoming flatter and more irregular, and develop broad, frequently undulating cytoplasmic veils which attach to the underlying substrate (Figs. 23-30). After 2-3 days, many ce11s become elongated, with thicker cord-like extensions of cytoplasm which attach onto the glass (Fig. 27).

Earlier studies on macrophages by Albrecht et al. [1] and Warfel and E1berg [99] and more recent studies on human mononuclear cells [63], murine macrophages [60, 62, 75] (Figs. 31-43) and isolated hepatic reticuloendothelial cells [78] (Figs. 44-51) have shown that this ce11 type, particularly when activated, has prominent ridge-like profiles and ruffled membranes, with few microvilli. In these respects, circulat­ing monocytes are similar to different tissue macrophages.

Monocytes 9

Fig. 9. Mononuclear cells separated by Ficoll-Hypaque gradient centrifugation. The spherical cells with varying numbers of microvilli are lymphocytes while the larger more irregular cells with ridge-like profiles and ruffles are monocytes ( x 13,200)

10 Monocytes

Monocytes I I

Figs. 10 and II. Mononuclear cells separated by Ficoll-Hypaque gradient centrifugation. The monocytes have ruffled membranes and ridge-like profiles while lymphocytes show microvilli (x 6000, x 7800)

II

12 Monocytes

Figs. 12 and 13. Close-up photomicrographs of mononuclear cells (isolated from normal peripheral blood), with particularly well-developed ruffled membranes. Note that even when cells are prepared without prior fixation in cell suspension, ruffled membranes are prominent (x 10,400, x 15,600)

Monocytes 13

Figs. 14 and 15. Peripheral blood monocytes separated by Ficoll-Hypaque gradient centrifugation. Note the very well-developed, broad-based ruffled membranes and absence of microvilli ( x 8400, x 7000)

14

15

14 Monocytes

Figs. 16 and 17. Mononuclear cells obtained from lymph node cell suspensions (separated by Ficoll-Hypaque gradient centrifugation), resembling monocytes with well-developed ruffles (x 11,900)

Figs. 18 and 19. Mononuclear cells (isolated from the peripheral blood of patients ~ with monocytosis) with prominent ruffles (x 3600, x 7200)

Monocytes 15

16 Monocytes

Fig. 20. Monocyte from the peripheral blood, after incubation with polystyrene latex beads. The latex beads attach to the ruffled surface of the cell and are partially ensheathed by these folds of cytoplasm (x 12,400)

Monocytes 17

Figs. 21 and 22. Monocyte IgG-EA rosettes showing attachment of red blood cells onto the surface of the monocytes and phagocytosis of rosetting erythrocytes (x 11,000, x 8400)

21

22

18 Monocytes

Figs. 23- 30. Human mononuclear cells (isolated from normal peripheral blood) which have attached onto glass and spread. Note the broad undulating folds of cytoplasm attaching cells onto the substrate. Ridge-like profiles and ruffles are readily seen (Figs. 23-26). Some elongated cells with cord-like extensions attaching to the glass are evident (Fig. 27). When cells are incubated with formalinized RBC these are phago­cytosed by sleeves of cytoplasm (Figs. 28- 30)

Monocytes 19

20 Isolated Peritoneal Macrophages

Fig. 31. Typical rounded peritoneal macrophage with ridge-like profiles and ruffles which has not yet spread onto glass ( x 8000)

Fig. 32. Rounded peritoneal macrophages alongside a spreading macrophage with promi­nent rolled edges ( x 2900)

Fig. 33. Peritoneal macrophage with surface ridges in the process of attaching onto the glass ( x 7200)

Fig. 34. Flattened peritoneal macrophage with ridge-like profiles and prominent intracy­toplasmic spherules ( x 2900)

Isolated Peritoneal Macrophages 21

Fig. 35. Flattened spreading peritoneal macrophages with readily evident nucleus and a few surface ridges ( x 3000)

Figs. 36 and 37. Comparison of nonactivated attached peritoneal macro phages (Fig. 36, x 700) and activated macro phages which have more prominent surface microprojections (Fig. 37, x 1200)

35

36 37

22 Isolated Peritoneal Macrophages

Figs. 38 and 39. Close-up of the surface of macro phages with ridge-like profiles and rumes ( x 6100)

Fig. 40. Flattened peritoneal macrophage with relatively large "craters," probably repre­senting phagocytic vacuoles ( x 2900)

Fig. 41. Flattened ceJl with attached and phagocytosed latex spheres (x 2700)

Isolated Hepatic Reticuloendothelial Cells 23

Figs. 42 and 43. Flattened macrophage in Fig. 42 engulfing echinocytic erythrocytes ( x 3000) via large "craters." This process of erythrocyte phagocytosis is seen from close up in Fig. 43 (x 6700)

Figs. 44 and 45. Isolated hepatic non parenchymal cells obtained from the liver after pronase treatment of the liver. This cell type was the most frequent encountered and most probably represents the Kupffer cell. Note prominent ruffles (x 13,500)

(Cells of Figs. 44- 51 prepared by Dr. C. Hershko, Haematology Dept., Hadassah University Hospital, Jerusalem, Israel)

42 43

44 45

24 Isolated Hepatic Reticuloendothelial Cells

Figs. 46 and 47. Reticuloendothelial cells attached to glass with prominent ruffles (x 2500, x 2700)

Figs. 48 and 49. Hepatic reticuloendothelial cells with prominent ruffles. Latex beads are attached onto their surfaces and are seen to be in close contact with ruffles ( x 6300)

Granulocytes 25

Figs. 50 and 51 . Two reticuloendothelial cells with markedly altered surface architecture after phagocytosis of latex beads. Prominent surface ruffles and blebs are present (x 6700)

Granulocytes (Figs. 52- 61) show a more varied spectrum of surface morphology with a wider range of microprojections including ridge­like profiles, small ruffles and microvilli. Most granulocytes, and these are mainly neutrophils, have ridge-like profiles and small ruffles, while eosinophils (Fig. 61) and particularly basophils (Braylan, personal communication) may have varying numbers of microvilli. The latter may be misidentified as lymphocytes in mixed populations of leuko­cytes; Wetzel and co-workers [100], using a positive method for identi­fying the same cells under the light microscope and the SEM, have pointed out this difficulty as a potential source of error. Examination of a single case of marked eosinophilia [72] confirmed this observation (Figs. 60, 61). Fortunately, eosinophils and basophils are present in small numbers in the peripheral blood and are infrequently en­countered in enriched populations of lymphocytes separated by the Ficoll-Hypaque method. Accordingly, the error that they may intro­duce when studying enriched leukocyte populations under the SEM, is minimal. Granulocytes with ridge-like profiles and small ruffles may resemble monocytes and in mixed leukocyte populations it is not possible to distinguish between these two cell types with any degree of certainty, although monocytes often have much more promi­nent ruffles .

26 Granulocytes

Fig. 52. Low-power photograph of critical point-dried granulocytes mixed with erythro­cytes, obtained from a normal individual. Individual granulocytes vary in shape and have different types of microprojections. Ridge-like profiles and small ruffles are most frequently encountered (x 4800)

Granulocytes 27

Fig. 53. Critical point-dried granulocytes mixed with erythrocytes, obtained from a patient with granulocytosis. Individual granulocytes show a variety of surface micropro­jections. Ridge-like profiles and small ruffles are most frequently seen (x 4500)

28 Granulocytes

54

.... Figs. 54 and 55. Close-up photographs of granulocytes (from normal individuals and from others with granulocytosis) showing transverse ridge-like profiles and ruffles, which are sometimes polarized to one edge of the cell (x 7100)

Granulocytes 29

56

______ ____

Figs. 56 and 57. Close-up photographs of granulocytes (from normal individuals and from others with granulocytosis), showing transverse ridge-like profiles and ruffles, which are sometimes polarized to one edge of the cell (x 71 00)

30 Granulocytes

Fig. 58. Circulating granulocyte with prominent ridge-like profiles and peripheral ruffles (x 7100)

Fig. 59. Circulating granulocyte with a dome-shaped nuclear region covered with irregu­lar{!at ridge-like profiles and very well-developed basal and peripheral ruffles (x 14,400)

Granulocytes 31

60

__ '----' 61

Figs. 60 and 6l. Circulating eosinophilic leukocytes isolated from a patient with marked eosinophilia in which almost all cells in the peripheral blood were eosinophils (more than 95%). Note the variation in surface morphology. Most of the cells had transverse ridge-like profiles (Fig. 60) while others showed short microvilli (Fig. 61) (x 13,000, x 16,200)

32 Lymphocytes and Thymic Cells

2. Lymphocytes and Thymic Cells: Review of the Factors Affecting the Surface Morphology of Lymphocytes

When circulating peripheral blood lymphocytes are not fixed in cell suspension but harvested on various substrates prior to fixation, they display varying numbers offinger-like microvilli and ruffles are seldom seen [43, 44, 65-68]. In these earlier studies many lymphocytes showed relatively few microvilli and the percentage of these cells correlated well with the number of T-cells identified by immunologic techniques. The proportion of bone marrow (B)-derived cells correlated well with the percentage of cells displaying many surface microvilli [42, 43-44, 65-68]. However, cells with overlapping surface features were always encountered and were difficult to classify. Similar findings were en­countered by Lambertenghi-Deliliers et al. [40], Cohnen et al. [18] and Renau Piqueras et al. [86] in their studies on circulating lymphocytes, collected onto glass, porous membranes or cell monolayers without aspiration and filtration. Particularly convincing in this respect was the observation that almost all murine and human thymocytes [42, 44,60,65, 76] had very few surface microvilli (Figs. 75-84), irrespective of their mode of preparation for SEM. Accordingly, it was suggested that the lymphocytes with fewer microvilli may represent T-cells, while cells with multiple microvilli were more likely to be B-cells. However, because of the presence of cells with overlapping features and the possi­bility of misidentification under the SEM, it was suggested that the cells examined always be identified by parallel immunologic techniques.

Further support for the correlation described above was obtained from the study of cultured lymphoblastoid cell lines (all of B-origin) and the Molt-4 cell line (T -origin, derived from a patient with acute lymphoblastic leukemia). Both our studies [65-67] and those of Lin et al. [46], Nilsson and Ponten [58] and Fagraeus et al. [24a], showed that the Molt-4 T-cells most frequently had few microvilli while cul­tured B-cell lines showed a spectrum of surface morphology but were most frequently villous in nature. In addition, when E-rosettes (T­lymphocyte marker) and EAC-rosettes (B-lymphocyte marker) were studied [36, 43, 67], using different methods of cell collection and fixation, the majority of rosetting T-lymphocytes did not display multi­ple microvilli, while most of the rosetting B-lymphocytes in EAC rosettes had moderate to markedly villous surfaces (Figs. 85-91). A larger number of villous rosetting T -lymphocytes were encountered in these samples, while about 5-10% of rosetting B-lymphocytes had smoother surfaces. Thus, from these studies, it was clear that in most cases, it was not possible to distinguish rosetting B- and T-lymphocytes with consistency particularly since it was apparent that the surface of the rosetting lymphocytes altered during this procedure [45, 67]. Similar results for rosetting lymphocytes were obtained by Jerrels and Hinrichs

Lymphocytes and Thymic Cells 33

[36]; however, Kay et al. [37] studying activated T-rosettes and Newell et al. [57] found no difference in rosetting and nonrosetting lymphocyte populations. These discrepancies were no doubt due to the different rosetting techniques employed and to variations in the mode of cell preparation and fixation used in the different studies [36, 37, 45, 57, 67). Accordingly, the presence of "smoother" rosetting T-cells in the above studies could not be attributed to the use of aspiration and filtration alone, as this technique 'was not employed by Lin et al. [45] and J errels and Hinrichs [36] in their studies.

Nevertheless, the above findings relating to differences in circulating B- and T-Iymphocytes must be reappraised in the light of recent studies by Alexander and co-workers [2,3] and Newell et al. [57] who have shown that almost all lymphocytes fixed in suspension prior to collec­tion onto the substrate have microvilli and that it is not possible to recognize subpopulations of circulating lymphocytes or distinguish between B- and T -cells using the SEM, when cells are fixed in suspension (Figs. 68-70). In addition, Alexander et al. [3] have shown that collec­tion of lymphocytes which have not been fixed in suspension, onto porous membranes, using filtration and aspiration, results in a loss of microvilli and that the number of "smoother" lymphocytes can be altered readily with variations in the technique, suggesting that all lymphocytes are villous and that their absence is an artifact. In this respect it is of interest to note that other workers [7, 18, 38, 43, 86, 98] have observed substantial proportions of smoother lympho­cytes in their studies in which cells were sometimes fixed in suspension or in vivo and collected without filtration and aspiration onto glass or cell monolayers. Accordingly, techniques cannot always be evoked to explain all the differences seen in the various studies and it is most likely that both external and in vivo microenvironmental factors are also of great importance.

Loor and Britt Hagg [49] studying the modulation of microprojec­tions on the lymphocyte surface and the redistribution of membrane­bound ligands with immunofluorescence microscopy, showed that mic­rovilli are very labile structures which are strongly modulated by environ­mental conditions and do not always seem to depend on the lymphocyte source. Thus the number of surface microvilli present may not be constant at any given time and it would be unwise to use it as a consistent and reliable criterion for establishing lymphocyte origin. Van Ewijk and co-workers [98] have demonstrated this elegantly in their studies on homing and recirculation of lymphocyte populations. They showed that both B- and T -cells appear to lack microvilli in their microenviron­ment in the spleen and lymph nodes, but gain them during recircula­tion. Contact with endothelium was by means of microvilli while passage through the endothelium occurred via withdrawal of micro­villi. In their study of thymic lymphocytes, K wock et al. (39a) showed

34 Lymphocytes and Thymic Cells

that contact with nylon fibers was sufficient for cells to develope microvilli. Recently Kay [38] has suggested that the surface morphol­ogy of some lymphocytes may depend on their stage of activation while Baur et al. [7] in their reappraisal of lymphocyte classification implied that the surface morphology may relate to the stage of B­and T-cell differentiation.

Despite the above-mentioned findings, it is noteworthy that in contrast to circulating lymphocytes, thymic lymphocytes [42, 44, 60, 65, 76] and cultured T -cells [8, 22, 24, 45, 58] fixed in suspension, still display relatively few microvilli, resembling the lymphocytes seen in lymph nodes in vivo [79, 98]. Thus, it does not appear that all lymphocytes always have multiple microvilli when fixed in suspension. On the other hand, it is clear from all the above studies, that microvilli on circulating lymphocytes are dynamic, labile structures which may vary in size and appearance in relation to a number of stimuli. Circulat­ing blood lymphocytes (perhaps preferentially T -cells), tend to lose their microvilli quickly if they are not fixed in suspension.

It should also be mentioned at this point, that when viable or fixed lymphocytes are examined by other modes of microscopy includ­ing interference contrast (No mar ski) optical microscopy [22, 87, 91, 93, 97], Hoffman modulation contrast microscopy [61], transmission and immunoelectron microscopy [29, 31, 86, 88] and cryofracture [86], it is possible to detect subpopulations of villous and nonvillous lym­phocytes, frequently in similar proportions to the number of B- and T -cell populations in the blood. Furthermore, from immunoelectron­microscopic studies, it seems that lymphocytes with fewer microvilli are most frequently T-derived [88]. These observations provide strong evidence in favor of the fact that lymphocytes with few microvilli are present in the peripheral circulation and that the lack of microvilli on lymphocytes is not always necessarily an artifact induced by the preparatory techniques used in SEM.

Lymphocytes 35

Figs. 62 and 63. Cryofractured human lymphocytes from the peripheral blood showing that some cells have multiple microvilli while others have few ( x 10,000, x 22,800). Published through the courtesy of Drs. J. Renau Piqueras, A. Martinez-Ramon, G. For­teza-Bover, Valencia, Spain

36 Lymphocytes

Fig. 64. Normal peripheral blood lymphocytes, not fixed in cell suspension, collected by aspiration and .filtration onto silver membranes. Cells with multiple microvilli are seen alongside others with multiple microvilli. Many platelets are also seen ( x 5400). This technique has been shown to produce loss of lymphocyte microvilli (as seen by the presence of smooth cells free of microvilli) (2, 3) and is not recommended for use in SEM

Lymphocytes 37

Figs. 65 and 66. Close-up of peripheral blood lymphocytes, not fixed in suspension, showing varying numbers of microvilli (x 15,600, x 16,800)

oS

66

38 Lymphocytes

6

Fig. 67. Close-up of a pe­ripheral blood lymphocyte with multiple microvilli. Cells were not fixed in sus­pension prior to collection onto the substrate (x 23,400)

Fig. 68. Lymphocytes, fixed in suspension, show­ing cells with varying numbers of microvilli. Most cells fixed in this fashion have microvilli (x 75(0)

Lymphocytes 39

Figs. 69 and 70. Typical close-up appearance of lymphocytes fixed in cell suspension showing multiple-microvilli of varying lengths (x 16,100)

69

70

40 Lymphocytes

Fig. 71. Peripheral blood lymphocytes from the buffy coat of a patient (not separated by gradient centrifugation and not fixed in cell suspension) showing microvilli (x 14,000)

Lymphocytes 41

Figs. 72 and 73. Peripheral blood lymphocytes (after separation and concentration) obtained from a patient with benign hypergammaglobulinemia. Cells were not fixed in suspension and the filtration-aspiration technique was used. Note the presence of multiple microvilli and the variation in their length. Some have a spiky appearance ( x 8900)

73

42 Lymphocytes

Fig. 74. Close-up of peripheral blood lymphocyte (obtained from a patient with hyper­gammaglobulinemia, shown in part in Fig. 72) with spiky, erect microvilli (x 29,600)

Figs. 75 and 76. Human and murine thymic lymphocytes, not fixed in cell suspension, showing relatively few microvilli (x 4500, x 8400)

44 Thymic Lymphocytes

Figs. 77 and 78. Thymic lymphocytes (fixed in suspension with 1% glutaraldehyde prior to collection onto substrate) showing relatively few microvilli (x 3800, x 7200)

Figs. 79 and 80. Thymic lymphocytes (fixed in suspension with 3% glutaraldehyde) showing irregular surfaces with relatively few microvilli. Note slight distortion of cells ( x ~ 100, x 6200)

Thymic Lymphocytes 45

Figs. 81 and 82. Close-up of thymic lymphocytes fixed in cell suspension, showing rela­tively few microvilli (x 12,800)

Fig. 83. Thymic lymphocyte-SRBC rosette (E-rosette ). Some erythrocytes have developed microvilli during rosetting (x 14,500)

46 Thymic Lymphocytes

Fig. 84. Thymic lymphocyte-SRBC rosette (E-rosette ) . Some spheroechinocytic erythro­cytes have developed elongated microvilli during rosetting, while the central thymic cell has also developed more microvilli (x 13,600)

Spontaneous T-Lymphocyte-Sheep Erythrocyte Rosettes 47

Fig. 85. T-lymphocyte- sheep erythrocyte rosette. The central rosetting lymphocyte has a moderate number of microvili and is surrounded by spheroechinocytic erythrocytes which show" point to poind" attachments (x 14,000)

48 Spontaneous T-Lymphocyte-Sheep Erythrocyte Rosettes

Fig. 86. T-Iymphocyte-sheep erythrocyte rosettes. The central lymphocyte has a moderate number of microvilli while the surrounding spheroechinocytic erythrocytes show typical "point" attachment to the cell surface

Spontaneous T-Lymphocyte-Sheep Erythrocyte Rosettes 49

Fig. 87. T-Iymphocyte-sheep erythrocyte rosettes. The rosetting T-cell shows the rarer type of broad zone attachment of erythrocytes to the lymphocyte surface. Perhaps this is related to the fact that the erythrocytes have not developed microprojections (x 11,200)

50 Spontaneous T-Lymphocyte-Sheep Erythrocyte Rosettes

Fig. 88. Central rosetting T -lymphocyte, covered with multiple microvilli and surrounded by a large number of echinocytic erythrocytes which show multiple microprojections and attach to the lymphocyte surface in "point to point" fashion (x 13,200)

Human B-Lymphocyte-EAC Rosettes 51

Fig. 89. B-Iymphocyte with moderate numbers of microvilli showing typical broad zone of RBC attachment to the lymphocyte (x 13,200)

Fig. 90. B-Iymphocyte with microvilli surrounded by sphereochinocytes. In this case, areas of "point to point" contacts are seen (at lower right of the rosette) between RBC and lymphocyte (x 12,200)

52 Human B-Lymphocyte-EAC Rosettes

Fig. 91 . Villous B-Iymphocyte surrounded by RBC. Note the broad zone of attachment between the lower RBC and the lymphocyte (x 14,000)

D. Labeling Studies on Lymphocytes

A number of SEM studies of labeled human and murine lymphocytes have been reported recently [32, 38, 47, 48, 55, 56] using hybrid anti­body techniques which employ tobacco mosaic virus (TMV) or latex spheres as markers. In these studies, lymphocytes were seen to have varying numbers of microvilli and almost no cells with ruffles were labeled. When cells were labeled with antitheta (8) antibody and TMV, the hybrid antibody attached predominantly to cells with few microvilli while the hybrid antibody prepared against B-cells attached mostly, but not in all cases, to villous cells [32] and quite frequently to micro­villi. Similar results were described by Lipscomb et al. [48] in their study. However, labeled overlapping populations were seen in the above studies and in those of Nemanic [56], Molday [55] and co­workers and Linthicum and Sell [47], and it was not possible to dis­tinguish different subpopulations on the basis of these results. How­ever, it does appear that the surface immunoglobulin is located in nonrandom areas [47, 48], frequently on the microvilli [47] and not on the smoother portions of the cell. Most of the above studies employed one marker which restricted visualization to only one type of receptor. Recently, Kay [38] has described new labeling techniques for SEM using three markers, each with a distinctive size and shape (KLH, SV40 and the T rbacteriophage), which will no doubt be most useful in the future.

It should, however, be remembered [38] that these SEM studies are difficult to perform and interpret. The marker sometimes obscures visualization of the underlying surface detail and prevents accurate quantitation and precise localization of the receptors. In addition, the antibody can also adhere nonspecifically to many surfaces and dissociate from the marker making interpretation difficult in some cases. Furthermore, the marker may enter the cell, bind in layers or be removed during preparatory procedures and in critical point­drying.

54 Murine Lymphocytes Labeled Using Tobacco Mosaic Virus (TMV) as a Label

Fig. 92. Most cells are diffusely labeled with TMV ; while some of the TMV are curled, others are more erect and some have collapsed on the cell surface ( x 10,600) (Hybrid antibody prepared and experiments performed by Dr. U. Hammerling, Memorial Sloan-Kettering Cancer Center, New York) (Figs. 92-101)

Murine Lymphocytes Labeled to Show Thy-I Antigen on Lymphocytes 55

Fig. 93. Cells with relative­ly rew microvilli show scanty labeling with clus­ters or TM V ( x 10.4(0)

Fig. 94. Murine lymph node cells labeled diffusely with TMV (x 10.(00)

97

56 Murine Lymphocytes Labeled Using Tobacco Mosaic Virus (TMV) as a' Label

Figs. 95 and 96. Close-up of murine lymph node cells labeled with TMV. Figure 95 shows dense labeling of cell with few microvilli (x 12,000), while Fig. 96 shows clusters of TMV adjacent to some microvilli (x 9800)

Fig. 97. Labeled cell showing the differences in size between TMV and microvilli which are easily distinguished from one another (x 14,000)

Murine Lymphocytes Labeled to Show Thy-I Antigen on Lymphocytes 57

Fig. 98. A more villous lymphocyte which shows only scanty virus label (x 14,000)

Fig. 99. Close-up of clusters of TMV easily distinguished from broader microvilli (x 37,800)

58 Murine Lymphocytes Labeled Using Tobacco Mosaic Virus (TMV) as a Label

Figs. 100 and IOJ. Close-up of clusters of TMV easily distinguished from microvilli (x 37,800, x 45,000). (In Fig. 100 the lymphocytes are labeled with hybrid antibody to show H2 antigen on lymphocytes)

Murine Lymphocytes Labeled to Show Ia Antigen on Lymphocytes 59

Fig. 102. Only one of the five lymph node cells is densely labeled with TMV. Although many lymphocytes with few microvilli did not label with this B-cell marker, occasional labeled cells with non villous surfaces were observed (x 11,200)

60 Murine Lymphocytes Labeled Using Tobacco Mosaic Virus (TMV) as a Label

Fig. 103. Lymphocyte with moderate numbers of microvilli labeled with clusters of TMV; some label is present on microvilli or near the base of microvilli (x 21,600)

Murine Lymphocytes Labeled to Show Ia Antigen on Lymphocytes 61

Fig. 104. Villous B-Iymphocyte densely labeled with TMV ( x 24,000).

(Hybrid antibody prepared and experiments performed by Dr. U. Hammerling, Memorial

Sloan-Kettering Cancer Center, New York) (Figs . 92- 101)

E. Other Factors Influencing the Surface Morphology of Lymphocytes and Mammalian Cells

We have mentioned earlier the importance of the effect of cell prepara­tion, collection and fixation [2, 3; 6, 101] on the surface morphology oflymphocytes. However, other factors also playa role and it has been shown that cell density and intercellular contact [89], and cell spread­ing [23 a], alter the surface morphology. Contact between rosetting lymphocytes and erythrocytes results in the appearance of microvilli on the lymphocytes and even on the rosetting red cells [45, 67, 68]. In these studies, smooth thymic cells [67] and cultured T -cells [45] become markedly villous during rosette procedures and after contact with nylon fibers [39 a].

Lin et al. [46] demonstrated the importance of temperature in this respect showing that extremes of temperature result in loss of microvilli which return when the cells are placed at room temperature or 37°C (optimal temperatures for processing ceHs for SEM).

In cultured cells Porter and co-workers [83] have shown that there are changes in the surface topography during the different phases of the cell cycle. These cells, however, were nonlymphoid in nature. In some cells microvilli are more frequently observed during the GI phase of the cell cycle while ruffles are more often seen in the S and G2 phases.

In viral infected, transformed and malignant cells, surface features are different from those of their normal nontransformed counterparts [4, 23, 25, 30, 33, 50, 84, 85]. In viral infetted cells, it appears that the initial alterations in the surface structure are not related to the cell cycle [30] and may be dependent on neoprotein synthesis [4, 25] in some cases. While it has been shown that virus infection per se may alter the surface structure of some cultured cells, this still remains to be studied in circulating human lymphocytes.

F. Mitogen-Transformed Lymphocytes

When normal circulating lymphocytes and thymic lymphocytes are cultured with phytomitogens for up to 72 h, a large proportion of cells are transformed to blasts. Phytohemagglutin (PHA), concanava­lin A (Con A) and pokeweed mitogen (PWM) result in blast transfor­mation of lymphocytes which develop different surface features than their nontransformed counterparts (examined before or after incuba­tion for 72 h without the addition of mitogens) [70, 96]. Peripheral blood lymphocytes transformed by PHA, Con A or PWM showed a variety of surface microprojections [70, 96]. These were most fre­quently finger-like or cone-shaped microvilli but occasional cells showed ruffled membranes and others had elongated uropods termi­nating in tuft-like extremities. Smooth thymic cells transformed by Con A become markedly villous [76] in nature but still maintain their T -cell markers [96], while nontransformed cells remain spherical in shape with relatively few microvilli.

Until now it has not been possible to distinguish the different blast types under the SEM on the basis of their surface morphology despite the fact that they retain their original surface markers when examined by immunologic methods.

Thymic Lymphocytes Transformed by Concanavalin A 65

Figs. 105 and 106. Typical surface features of thymic lymphocytes, fixed in suspension in I % glutaraldehyde, prior to contact with concanavalin A (x 13,500)

Figs. 107 and 108. Two typical concanavalin A-transformed thymic lymphocytes showing multiple microvilli which develop during the process of transformation, 72 h after contact with the mitogen ( x 13,500)

66 Mitogen-Transformed Lymphocytes

Fig. 109. Close-up of pokeweed mitogen (PWM) transformed lymphocyte, 72 h after incubation with the mitogen ( x 14,000)

Mitogen-Transformed Lymphocytes 67

Figs. llO-113. Phytohaemagglutinin (PHA)-transformed lymphocytes, 24-72 h after in­cubation with the mitogen. Most cells are villous in nature and display short stub-like or longer" spiky" microvilli (Fig. 110, x 14,000; Fig. III , x 14,000 ; Fig. 112, x 34,500; Fig. 113, x 14,000)

110

III

Legends see page 67

Mitogen-Transformed Lymphocytes 69

Fig. 114. Mitogen-transformed cell with relatively few microvilli (x 14,000)

70 Mitogen-Transformed Lymphocytes

11 5 __ ,-

11 6

Figs. 115- 118. Mitogen-transformed cells with large peripheral ruffles which are some­times well developed. These are less frequently seen with PHA, Con A or PWM but are often seen in PM A-transformed cells (x 12,000- 15,000)

Mitogen-Transformed Lymphocytes 71

117

72 Mitogen-Transformed Lymphocytes

119

120

Figs. 119- 124. PHA-transformed cells showing well-developed uropod formation. Note the typical tufted extremities of the uropod covered with microvilli and flipodia (Fig. 119, x 6200 ; Fig. 120, x 12,600 ; Fig. 121 , x 14,000 ; Fig. 122, x 28,000); Figs. 123 and 124 (x 15,600) show uropods which are slightly different in appearance than those seen in earlier figures

Mitogen-Transformed Lymphocytes 73

74 Mitogen-Transformed Lymphocytes

Legend see page 72

Mitogen-Transformed Lymphocytes 75

124

Legend see page 72

G. Cultured Cell Lines

A variety of lymphoblastoid, lymphoma, "histiocytic", and myeloma cell lines have been studied under the -SEM recently [8, 22, 24a, 39, 41,58,59,65,94]. While lymphoblastoid cell lines derived from various sources (patients with infectious mononucleosis, lymphoma or normal donors) show no specific malignant characteristics and are most likely to be derived from normal nonmalignant cells, Lymphoma cell lines derived from patients with African and non-African Burkitt's lym­phoma have different growth characteristics and can be recognized in many cases to be derived from the original malignant lymphoma cells [58]. All these cell lines are of the B-Iymphocyte type and bear B-cell markers such as, surface immunoglobulin, receptors for comple­ment or Epstein-Barr virus. As in earlier SEM studies [65-67], the B-cell lines showed a spectrum of surface morphology ranging from villous to nonvillous in nature, but the vast majority, particularly in the case of Burkitt's lymphoma, are villous in type. Relatively few cells with surface blebs, ruffles or uropods are seen in the lymphoma cell lines. Other, apparently nonlymphomatous lymphoblastoid cell lines, showed more variation in shape and size. The majority of lym­phoblastoid cells were spherical with varying numbers of microvilli but more hand-mirror forms were evident and ridge-like profiles and blebs were more frequently encountered. Nilsson and Ponten [58] have shown that the above differences probably relate to the fact that lymphoblastoid cells show more directional locomotion than lym­phoma cells which display surface movement but do not show direc­tional locomotion of the entire cell. The leukemic Molt-4 cell line, known to be T -derived, was distinctly different in that the cells displayed fewer surface microvilli. The vast majority of cells were of the smooth type, irrespective of their mode of preparation and fixation for SEM. However, more cultured T-cell lines will have to be studied in order to establish whether the apparent differences be­tween cultured B-cells and the Molt-4 T-cell line described in earlier studies [65-67] are consistently encountered.

Cultured human and murine myeloma cells have varying numbers of microvilli, but characteristically display multiple surface blebs of varying size [41,58,59,81]. The presence of surface blebs is a consistent finding in myeloma cells and does not appear to be a function of the cell cycle [41]. Blebsare also found on nonproducer myeloma cells [41,59,81]. Similar surface features are also encountered on malignant plasma cells obtained from patients with plasma cell leukemia [81].

Cultured Cell Lines 77

A few "histiocytic" cell lines, derived from patients with so-called histiocytic lymphoma have been examined recently under the SEM [94, 39]. In some cases the majority of cells showed surface features similar to those described for monocytic/histiocytic cells, i.e., promi­nent lamellae and ruffles and not microvilli. In these cases the apparent nonlymphoid character of the cells was confirmed by electron micro­scopy and cytochemistry and it is indeed possible that these are true histiocytic cell lines [39, 94]. In other cases the so-called histiocytic cells had multiple microvilli resembling lymphoid cells [39].

78 Cultured Lymphoblastoid, Lymphoma, Myeloma and "Histiocytic" Cell Lines

Fig. 125. Lymphoblastoid cells (not fixed in cell suspension), showing variation in shape and type of microprojections seen. Some cells have microvilli while others show periph­eral ruffles ( x 5600)

Fig. 126. Typical example of a spherical Iymphoblastoid cell with finger-like microvilli of varying length (x 7200)

Fig. 127. Spherical cultured "Iymphoblastoid" cell with multiple, particularly long, well-developed microvilli (x 8400)

80 Cultured Lymphoblastoid, Lymphoma, Myeloma and "Histiocytic" Cell Lines

Fig. 128. Three spherical Iymphoblastoid cells showing varIatIOns in the shape and type of surface microprojections encountered in these cells. Cells show microvilli and well-developed peripheral rumes - perhaps associated with directional locomotion (x 6500)

Cultured Lymphoblastoid Cell Lines 81

Fig. 129. Irregular Iymphoblastoid cell with well-developed broad based marginal rumes (x 12,000)

82 Cultured Lymphoblastoid, Lymphoma, Myeloma and "Histiocytic " Cell Lines

Fig. 130. Irregular Iymphoblastoid cell with small stub-like microvilli , peripheral ruffles and a few blebs (x 16,000)

Cultured Lymphoblastoid Cell Lines 83

Fig. 131. Spherical Iymphoblastoid cell with microvilli, a few blebs and exaggerated peripheral broad-based ruffles (x 15,000)

132

84 Cultured Lymphoblastoid, Lymphoma, Myeloma and "Histiocytic" Cell Lines

Figs. 132 and 133. The figures show the typical surface features of Burkitt cells. Most cells show varying numbers of short microvilli. Blebs, ruffles and uropods are infre­quently encountered (Fig. 132, x 7700; Fig. 133, x 9400)

Cultured Burkitt Lymphoma Cells 85

133

86 Cultured Lymphoblastoid, Lymphoma, Myeloma and "Histiocytic" Cell Lines

Figs. 134 and 135. The figures show the typical surface features of Burkitt cells. Most cells show varying numbers of short microvilli. Blebs, ruffles and uropods are infre­quently encountered ( x 18,000; x 16,000)

Cultured Burkitt Lymphoma Cells 87

135

88 Cultured Lymphoblastoid, Lymphoma, Myeloma and "Histiocytic" Cell Lines

Figs. 136- 139. Typical surface features of cultured Molt 4 cells. Most cells have few microvilli (x 8000; x 7000). In some more rapidly growing cultures cells with larger numbers of microvilli are more frequently seen as illustrated in Figs. 139 and 140 (x 10,200 ; x4100)

Figs. 140- 143. Molt 4 cells rosetted with sheep erythrocytes (E-rosettes). Rosetting cells are more villous than nonrosetting cells, indicating that the rosette procedure may alter the surface of the cell

Molt-4 Cell Line 89

Figs. 140 and 141. Close-up of different rosetting cells and illustrating the point-to-point contact so typical of E-rosettes ( x 8900; x 10,300)

140

141

90 Cultured Lymphoblastoid, Lymphoma, Myeloma and "Histiocytic" Cell Lines

142

Figs. 142 and 143. Close-up of different rosetting cells illustrating the point-to-point contact so typical of E-rosettes ( x 10,300; x 28,000)

Fig. 144. Villous surfaces of an IgG myeloma cell variant which is a non producer ; some surface blebs are present ( x 6300)

Fig. 145. Close-up of another nonproducer myeloma cell with some bleb formation (x 5800)

Fig. 146. IgO Myeloma producer cells with multiple surface blebs (x 4500)

144

145 146

92 Cultured Lymphoblastoid, Lymphoma, Myeloma and " Histiocytic " Cell Lines

Fig. 147. Close-up of IgG myeloma producer cell showing multiple blebs (x 15,000)

Cultured Myeloma Cells 93

Fig. 148. Close-up of IgG myeloma producer cell showing mUltiple blebs (x 12,600)

Figs. 149 and 150. High magnification of surface blebs on myeloma cells (x 12,600)

14

149 150

94 Cultured Lymphoblastoid, Lymphoma, Myeloma and "Histiocytic" Cell Lines

Figs. 151- 154. Some of the "histiocytic" cell lines showed a majority of cells which have well-developed ruffles, flattened ridge-like profiles and bleb-like excrescences

( x 5600-6400)

Cultured "H istiocytic" Cell Lines 9 5

Fig. 155. Cultured "histiocytic" cell with marked bleb formation (x 5600)

Fig. 156. Other " histiocytic" cell lines showed a predominance of cells displaying multi­ple microvilli, resembling cultured lymphoid cells ( x 7200)

155 156

96 Cultured K-562 Myeloid Cell Line

Figs. 157 and 158. K-562 "myeloid " cells have relatively few microprojections. Small rumes and transverse ridge-like profiles were seen on some cells (x 7700)

H. Surface Features of Leukemic Cells

During the past 4 years we have studied cells from approximately 200 cases of acute and chronic leukemia under the SEM [69, 71, 73, 80]. In most of the cases, cells were prepared for SEM without prior fixation in suspension and only a minority of cases were prepared for SEM using fixation in cell suspension. Cases studied included close to 100 patients with chronic lymphocytic leukemia (CLL) and lymphoma in the leukemic phase (LSL), 26 cases of acute lymphoblas­tic leukemia (ALL), 10 cases of "hairy" cell leukemia (leukemic reticuloendotheliosis, LRE) 5 cases of Sezary syndrome [77] and 2 cases of plasma cell leukemia [81]. The remaining patients with non­lymphoblastic leukemia included cases of acute and chronic myeloid leukemia, myelomonocytic, monoblastic, "histiocytic" and undifferenti­ated leukemia. In these cases the overall population of nonlymphocytic leukemic cells, i.e., myelomonocytic and histiocytic cells, could, in most instances, be distinguished from the majority ofleukemic lymphoid cells, on the basis of their surface ultrastructure. Despite the fact that a spec­trum of surface morphology was present, it was possible in many instances to classify cases according to the dominant cell type seen. Thus, leukemic monoblasts, and histiocytes resembled normal monocytes and macro phages and displayed well-developed broad-based ruffles and prominent ridge-like profiles [12, 71, 73]. Most leukemic myeloid cells had raised ridge-like profiles and small ruffles while a minority of cells showed microvilli. Patients with myelomonocytic leukemia had mixed populations of cells showing ruffles and ridge-like profiles. Many of the leukemic monoblasts and histiocytes appeared to have better developed broader ruffles and more prominent ridge-like profiles than their normal counterparts.

Circulating cells from patients with CLL, LSL and ALL differed from myelomonocytic cells and showed varying numbers of finger-like microvilli (Figs. 188 - 226), while ruffles and ridge-like profiles were less commonly encountered in these cases. In cases of CLL, cells with a spectrum of surface morphology were seen and varying numbers of microvilli were seen. Nevertheless, cells with moderate to mark­edly villous surfaces were most frequently observed, while in about 15 % of cases, a fairly large proportion of cells with few surface micro­villi were seen. In another 12 cases, cells with few surface microvilli dominated. In the cases of CLL, examined after cells were prepared by fixation in cell suspension, a similar spectrum of surface morphol­ogy was seen as described above. Nevertheless, more cases will have

98 Surface Features of Leukemic Cells

to be studied after fixation in cell suspension in order to compare these two techniques. Almost all of these cases of CLL including those with few surface microvilli were of the B-type when studied by immunologic methods. No accurate attempt was made to correlate the surface structure as seen unter the SEM with the stage of differenti­ation or degree of maturation of the leukemic B-cells as established by immunologic methods, i.e., with the degree of immunofluorescence encountered using fluorescent antisera. The majority of LSL cases were of the villous type but in a proportion of them, cells with few microvilli dominated. CLL and LSL cells studied by other groups (Mathe et al. [51], Catovsky et al. [14], Cohnen et al. [18], and Lamber­tenghi-De1iliers et al. [40]) showed similar features. It is of interest to record that in other cases, cells with fine ridge-like profiles dominated. These cases, also encountered by Golomb et al. [27 a] in their reported series, may represent subtypes of lymphocytic leukemia which need to be studied more carefully, and are indeed difficult to distinguish from other nonlymphoid leukemic cells which they resemble (Figs. 201-207). In rare instances cells with multiple surface blebs were seen. These cells resembled leukemic plasma cells and myeloma cells [41, 81].

In the few cases of Sezary syndrome examined [26, 73, 77], the abnormal circulating Sezary cells of the T-type, showed varying numbers of finger-like microvilli and many moderate to markedly villous cells were seen. A proportion of Sezary cells were irregular with clusters of microvilli polarized to one end of the cell. Some cells showed a tapered portion of cytoplasm at their peripheries resem­bling small uropod-like structures [77]. However, cells did not display other types of microprojections like ruffles frequently and in this re­spect were similar to other leukemic lymphocytes. Despite the fact that most CLL and LSL cells were of the villous type. it was not possible to distinguish leukemic B- and T-cells from one another or from cells which bore both B- and T-cell markers on the same cell, on the basis of their surface structure [69, 73, 92]. Similar findings have also been described by Reyes [88] and Gourdin [29] and co­workers using immunoelectromicroscopy.

In contrast to the above cases of CLL and LSL, in most of the 26 cases of ALL and in both cases of acute undifferentiated leukemia, the dominant circulating leukemic cell had few surface microvilli, irre­spective of the mode of cell preparation and fixation used [73, 80]. However, rarer cases of ALL with a majority of villous cells have been encountered in our studies [73, 80] and by others [14, 35a]. Recently Coleman et al. [19] described similar findings in cells collected onto glass in 27 cases of childhood ALL studied under the SEM. In all their cases cells with few microvilli dominated. This suggests that many of the less mature undifferentiated lymphoblasts of ALL (which are" null" type in close to 75% of cases and T-derived in

Surface Features of Leukemic Cells 99

approximately 25% of cases) have a different surface morphology than the more differentiated leukemic B-cells of CLL.

The" hairy" cells of leukemic reticuloendotheliosis are of particu­lar interest as there is still much controversy concerning their true nature and derivation [13, 20, 35]. Some authors have claimed that these cells are histiocytes [20] while others have shown them to be lymphocytes of B-derivation [13], which are capable of phagocytosis. Their surface morphology is interesting [27] as many cells resemble monocytes or histiocytes, display prominent ruffles and ridge-like profiles and phagocytose latex particles. However, clusters of stub-like microvilli are also seen on these cells while others are hybrid-like in appearance and show ruffles and microvilli in polar "distribution and in approximately equal numbers. It is indeed possible that the ruffles present on these "lymphoid" cells, like those on monocytes and leukocytes, enable them to phagocytose. Under the SEM it is relatively easy to distinguish these cells from CLL cells on the basis of their surface morphology.

1 00 Leukemic Cells

159

160

Figs. 159 and 160. Low-power photographs showing the overall populations of cells, most of which have transverse ridge-like profiles or ruffles and few microvilli . Some cells have relatively few microvilli. Some cells have relatively few microprojections ( x 4300)

Myeloblastic and Myelomonocytic Leukemia 101

Figs. 161 and 162. Fig. 161 shows buffy coat from a patient with chronic myeloid leukemia showing erythrocytes and leukemic cells with ridge-like profiles and small ruffles (x 4800). Fig. 162 shows close-up of one of the cells (x 12,000)

102 Leukemic Cells

Fig. 163. Buffy coat from a patient with acute myeloblastic leukemia showing erythrocytes and three leukemic cells with ridge-like profiles ( x 5600)

Myeloblastic and Myelomonocytic Leukemia 103

Figs. 164 and 165. Higher magnification of leukemic myeloblasts showing transverse ridge-like profiles and ruffles ( x 8600)

164

104 Leukemic Cells

Figs. 166-169. Close-up photographs of individual leukemic myeloblasls and promyelo­cyles from different cases (x 8000 x 14,000) showing ridge-like profiles and rumes

Myeloblastic and Myelomonocytic Leukemia 105

Figs. 170-173. Close-up photographs of individual leukemic myeloid cells from different cases showing ridge-like profiles and ruffles ( x 5400, x 7200)

170 171

172 173

106 Leukemic Cells

Figs. 174 and 175. Low-power photographs of leukemic monocytes and monoblasts show­ing that the majority of cells have well-developed ruffles and ridge-like profiles while few cells show multiple microvilli (x 4500, x 4700)

Monoblastic, Monocytic and" Histiocytic" Leukemias 1 07

Figs. I 76 and I 77. Lower-power photographs of leukemic monoblasts with well-devel­oped, broad ruffles. Fig. 177 shows a proportion of blasts with very few microprojections (x 4200)

176

177

108 Leukemic Cells

Figs. 178 and 179. Higher-power photographs of leukemic monoblasts and monocytes with prominent ruffles (x 8500, x 9100)

Mono blastic, Monocytic and "Histiocytic" Leukemias lO9

Figs. 180 and 181. Higher-power photographs of leukemic monoblasts and monocytes with prominent ruffles (x 8500, x 9100)

180

I I

Fig. 182. Close-up of individual leukemic monoblast showing very well-developed rumes and relative lack of microvilli (x 8400)

Fig. 183. Close-up of individual leukemic monoblast showing very well-developed rumes and relative lack of microvilli ( x 9800)

Monoblastic, Monocytic and" Histiocytic" Leukemias 111

Figs. 184--187. Close-up of individual leukemic monoblasts, monocytes and " histiocytes " showing very developed ruffles and lack of microvilli (x 5600, x 7200)

112 Leukemic Cells

Figs. 188 and 189. Groups of CLL cells, illustrating the spectrum of surface morphology commonly seen. The most frequent microprojections seen on these cells are microvilli. Cells most frequently showed moderate to markedly villous surfaces. In these photo­graphs cells with varying numbers of microvilli are seen alongside cells with fewer microvilli (x 10,000). Fig. 189 shows a group of moderately villous CLL cells

Chronic Lymphocytic Leukemia (CLL) 113

Figs. 190 and 191. Close-up photographs of moderate and markedly villous CLL cells (x 6400)

Figs. 192 and 193. CLL cells fixed in suspension showing multiple microvilli (x 6100; x 8600)

190 191

192 193

194

114 Leukemic Cells

Figs. 194 and 195. Transmission electron micrographs of CLL cells showing few micro­villi (Fig. 194, x 19,500) and multiple microvilli (Fig. 195, x 21,600) (Photographs taken by Janet Arce, New York)

Chronic Lymphocytic Leukemia (CLL) 115

Figs. 196 and 197. Overall view of CLL cells from a case in which the majority of cells had moderate to markedly villous surfaces ( x 4000)

196

197

116 Leukemic Cells

Figs. 198 and 199. Overall view of CLL cells from cases which showed a majority of cells with relatively few microvilli or a mixture of cells with few or multiple microvilli (x 4300)

Chronic Lymphocytic Leukemia (CLL) 117

Fig. 200. Overall view of CLL cells, some of which show microvilli and others flatter ridge-like profiles ( x 4800)

Fig. 201. Overall view of cells from a patient with eLL whose cells show flat ridge-like profiles ( x 4500)

201

118 Leukemic Cells

Figs. 202 and 203. eLL cells which show transverse ridge-like profiles and small ruffles (Fig. 202, x 4500). Close-up of some of these cells is seen in Fig. 203 (x 8000). Surface features in these cases are similar to those seen in monocytic cells

Chronic Lymphocytic Leukemia (CLL) 119

Figs. 204 and 205. Circulating cells from a patient with lymphocytic lymphoma in leukemic phase and another patient with eLL, which are slightly atypical in that they are more irregular and somewhat elongated with ridge-like profiles and small peripheral ruffles (Fig. 204, x 10,800 ; Fig. 205, x 4300)

120 Leukemic Cells

206

207 ........

Figs. 206 and 207. Circulating cells from a patient with lymphocytic lymphoma in leukemic phase and another patient with CLL, which are slightly atypical in that they are more irregular and somewhat elongated with ridge-like profiles and small peripheral ruffles (Fig. 206, x 4300 ; Fig. 207, x 9200)

Chronic lymphocytic leukemia (Cll) 121

Figs. 208 and 209. Overall view of cells from an unusual case of eLL which show some microvilli and multiple blebs ( x 4300)

208

209

122 Leukemic Cells

Fig. 210. Group of Sezarysyndrome (SS) cells, prefixed in suspension showing typical surface features encountered. Cells are mainly spherical, vary in size and display varying numbers of finger-like microvilli ( x 5200)

Fig. 211 . Group of irregular SS cells, fixed in suspension and collected onto a Millipore filter showing variation in size and shape and polarization of microvilli (x 5400)

Fig. 212. Two SS cells with moderate and markedly villous surfaces ( x 7900)

Fig. 213 . Close-up of SS cell with multiple microvilli ( x 13,500)

Sezary Syndrome (SS) Cells 123

Figs. 214 and 215. Close-up of two spherical SS cells with microvilli polarized to one end of the cell (x 12,200, x 9000)

Figs. 216 and 217. Close-up of irregular SS cells showing tendency of polarized microvilli to form small uropod-like structures at times ( x 9000)

2 18 219

220 221

124 Leukemic Cells

Figs. 218-221. Overall view and medium-power photographs of acute lymphoblastic leukemia (ALL) cells from different cases (some fixed in cell suspension and others not) showing typical surface features of these cells. Although villous cells are present, most cells have few microvilli (x 1700, x 4000)

Acute Lymphoblastic Leukemia (ALL) 125

Fig. 222. Higher magnification of ALL cells showing a group of ALL cells with varying numbers of microvilli ( x 5000)

126 Leukemic Cells

Figs. 223 and 224. Close-up of ALL cells with almost no microvilli (x 6300, x 7200)

Fig. 225. Close-up of ALL cells with relatively few microvilli (x 5400)

Fig. 226. Close-up of rarer example of more villous ALL cell (x 12,000)

"Hairy Cell" Leukemia (HCL) 127

Figs. 227 and 228. Lower-power photographs of buffy coats from two patients with "hairy cell" leukemia (HCL) in leukemic phase showing typical cells which bear both microvilli and ruffles ( x 4700)

128 Leukemic Cells

Fig. 229. Close-up of some "hairy cells " with prominent rumes. Some cells also have clusters of stub-like microvilli (x 10,400)

Fig. 230. " Hairy cell" with particularly long well-developed spiky ruffles (x 11,200)

"Hairy Cell" Leukemia (HCL) 129

Fig. 231. "Hairy cell" showing " hybrid-like" appearance with well-developed rumes at one end and microvilli at the other ( x 13,000)

232 233

234 235

130 Leukemic Cells

Figs. 232- 235. Close up of different" hairy cells" showing characteristic surface feature (x 6300)

"Hairy Cell " Leukemia (HCL) 131

Fig. 236. Photograph of "hairy cells" phagocytosing attached polystyrene latex beads The round lymphocytes with microvilli do not ingest the beads (x 10,900)

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Subject Index

Acute lymphoblastic leukemia 97, 98, 124--126

Air drying I, 2--4

Basophils 25 Blebs 76,98

on CLL cells 98, 121 on "histiocytic" cells 94,95 on lymphoblastoid cells 82 on myeloma cells 76,91-93,98 on plasma cells 76,91-93,98

Burkitt lymphoma 76, 84--87

Cell Cycle influence on surface morphology 63

Chronic lymphocytic leukemia (CLL) 97-99, Il2-121

Chronic myeloid leukemia (CML) 96, 97, 101

Concanavalin A 64, 65 Critical point drying (CPD) I, 5 Cryofracture of lymphocytes 34, 35 Cultured cell lines

Burkitt lymphoma 76, 84--87 "Histiocytic" lymphoma 77,94--95 K-562 myeloid line 96 Lymphoblastoid 32, 76, 78-83 Lymphoma 76, 97, 98 Molt 4 32, 34, 76, 88-90 Myeloma 76,91-93,98

EAC-rosettes 32, 33, 51-52, 63 E-rosettes 32, 33, 47-50, 63, 89-90

activated 33 Eosinophils 25, 31

Ficoll-Hypaque 5,25 Fixation 5, 6, 33 Freeze drying I

Granulocytes normal 8, 25-31 leukemic 97, 100-105

"Hairy" cell leukemia (Leukemic reti­culo-endotheliosis) 99, 127-131

Hepatic, reticuloendothelial cells 8, 23-25

"Histiocytes" 77, 94--95, 97 cultured 77, 94--95, leukemic 97, III

"Histiocytic" Lymphoma 77,94,95,97 Hybrid antibody techniques 53, 55--61

Bacteriophage 53 Latex 53 SV-40 53 Tobacco Mosaic Virus (TMV) 53,

55-61

Immunoelectronmicroscopy of lympho­cytes 34, 53, 55--61

Intercellular contact 63 Interference microscopy 34

Leukemias 97-99, 100-131 acute lymphoblastic (ALL) 97, 98,

124--126 chronic lymphocytic (CLL) 97,98,

112-121 chronic myeloid (CML) 97, 101 "Hairy" cell 99, 127-131 " Histiocytic" 97, III Monoblastic 97, lO6-Ill Monocytic 97, lO6-Ill Myeloblastic 97, 100-105 Myelomonocytic 97, 100-105 Promyelocytic 97, 104 Sezary Syndrome 98, 122-132

Leukemic reticuloendotheliosis (LRE) 99, 127-131

See "Hairy" cell leukemia Leukocytes 5--42

fractionation of 5

leukemic 97-99, 100-131 normal I, 5--42 preparation of 5

140 Subject Index

Lymphocytes 32-34, 35--42, 47-52 activation 34 air dried 1 bone marrow (B) derived 32, 33 contact with endothelium 33 cryofracture of 34--35 E-rosettes 32, 33, 47-50, 63 EAC-rosettes 32, 33, 51-52, 63 effect of cell preparation on 5, 6, 33,

34,63 effect of fixation on 5, 6, 33, 34, 63 interference microscopy of 34 immunoelectronmicroscopy of 34 labelling of 53, 54--61 leukemic 97,98,112-121 normal 32--42 mitogen transformation 63, 64, 66-75 modulation contrast microscopy of 34 peripheral blood 32--42 temperature 63 thymus-derived (T) 32, 33

Lymphoma Burkitt 76, 84-87

"Histiocytic" 77, 94-95 Lymphoma in leukemic phase 76,

97-98 Lymphoblastoid cells 32, 76, 78-83

Macrophages 8, 20-25

human 8 murine 8, 20-23 peritoneal 8, 20-23 reticuloendothelial 8, 23-25

Microenvironment, effects of 33, 34 Microscopy

Hoffman modulation contrast 34, immunoelectronmicroscopy 34, 53 immunofluorescence 34 interference contrast 34 phase contrast 34

Microvilli 1, 6, 32-34, 63, 64, 97-99 Mitogen transformation 64, 65-75 Molt 4 cells 32, 33, 76, 88-90 Monocytes

E-A rosettes 8, 17 leukemic 97, 106-111 normal 8,9-19 phagocytosis by 8, 16, 19 spreading 18, 19

Myeloblastic leukemia 97, 100-105 Myeloma 76,91-93, 98 Myelomonocytic leukemia 97, 100-105

Neutrophils 25 Null cells in ALL 98

Philadelphia Positive CML 96 Phytohemagglutinin 64, 67 Plasma cells 76, 98 Pokeweed mitogen 64, 66 Polylysine 6 Promyelocytic leukemia 97, 104

Reticuloendothelial cells from liver 8, 23-25

Ridge-like profiles (ridges) onCLLcells 98,117-120 on K-562 myeloid cells 96 on lymphoblastoid cells 76, 78 on normal granulocytes 8,25,26-31 on leukemic granulocytes 97, 100-105

Ruffles on"Hairy" cells 99, 127-131

on "Histiocytic" cells 77,94--95, 97, III

on leukemic monocytes 97, 106-111 on lymphoblastoid cells 76, 80-83 on macrophages 8, 20-25 on normal monocytes 8, 9-19

Sezary Syndrome 98, 122-123

Techniques 5, 6, 32-34 Temperature influence on surface

morphology 63 Thymic cells 32-34, 63-64

contact with nylon 33, 34, 63 human 32-34, 43--46 mitogen transformation of 64, 65 rosettes with SRBC 63, 64, 65-75

Transformed cells 63, 64, 65-75

Uropods on lymphoblastoid cells 76, 80 on mitogen transformed cells 64,

72-75 on Sezary cells 98, 123

Viral transformation of cells 63

H. Begemann, 1. Rastetter

Atlas of Clinical Haematology Initiated by L. Heilmeyer, H. Begemann With an Appendix on Tropical Diseases by W.Mohr Translated from the 2nd completely revised German edition by H. 1. Hirsch 191 figures in color and 17 in black and white. XV, 324 pages. 1972 ISBN 3-540-05949-0 Distribution rights for Japan: Maruzen Co. Ltd., Tokyo India: Nlied Publishers, New Delhi

M. Bessis

Blood Smears Reinterpreted Translated from the French by G. Brecher 342 figures mostly in color. XVI, 270 pages. 1977 ISBN 3-540-07206-3

M. Bessis

Corpuscles Atlas of Red Blood Cell Shapes 121 figures. 147 pages. 1974 ISBN 3-540-06375-7 Distribution rights for Japan: Maruzen Co. Ltd., Tokyo

M. Bessis

Living Blood Cells and their Ultrastructure Translated by R I. Weed 521 figures and 2 color plates. XXII, 767 pages. 1973 ISBN 3-540-05981-4 Distribution rights for Japan: Maruzen Co. Ltd., Tokyo

Experimental Hematology Today Editors: S.1. Baum, G. D. Ledney 147 figures. XVIII, 253 pages. 1977 ISBN 3-540-90208-2

International Symposium o/the Institutjiir Hiimatologie. GSF October 28- 30, 1976, Neuherberg/Munich

Immunological Diagnosis of Leukemias and Lymphomas Editors: S. Thierfelder, H. Rodt, E. Thiel 98 figures, 2 in color, 101 tables. X, 387 pages. 1977 ISBN 3-540-08216-6

Lymphocytes, Macrophages, and Cancer Editors: G.Mathe, I.F1orentin, M.-C.Simmler 53 figures. IX, 160 pages. 1976 (Recent Results in Cancer Research, Volume 56) ISBN 3-540-07902-5

D.Metcalf

Hemopoietic Colonies In Vitro Cloning of Normal and Leukemic Cells 54 figures, 28 tables. IX, 227 pages. 1977 (Recent Results in Cancer Research, Volume61) ISBN 3-540-08232-8

K Lennert

Malignant Lymphomas Other than Hodgkin's Disease Histology-Cytology-Ultrastructure­Immunology In collaboration with N. Mohri, H. Stein, E. Kaiserling, H.-K MUller-Hermelink 220 figures partly in color. Approx 800 pages. 1978 (Handbuch der speziellen pathologischen Anatomie und Histologie Band I, Tei13B) ISBN 3-540-08020-L

Red Cell Shape Physiology, Pathology, Ultrastructure Editors: M. Bessis, R I. Weed, P. F. Leblond Proceedings of a Symposium held at the Institute of Cell Pathology, Hopital de Bicetre, France, June 20-21, 1972 147 figures. VIII, 180 pages. 1973 ISBN 3-540-06257-2 Distribution rights for Japan: Maruzen Co. Ltd., Tokyo

Springer-Verlag Berlin Heidelberg New York

Subscription information: 1978, Volume 4 (3 issues)

North America: 1978: US $ 50.20, including postage and handling. Subscriptions are entered with prepayment only. Send your order or request to : Springer-Verlag New York Inc. 175 Fifth Avenue, New York, NY 10010, USA

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Sample pages available upon request.

Springer-Verlag Berlin Heidelberg New York

Blood Cells Editor-in-Chiif": Marcel Bessis, Institutde Pathologie Cellulaire, Hopital de Bicetre, F-94270 Kremlin-Bicetre, France

Senior Consulting Editors: Jean Bernard, Paris, France; George Brecher, San Francisco, Calif, USA

Editorial Board: D. F. Bainton, San Francisco, CA, USA; E. Beutler, Duarte, CA, USA; E. P. Cronkite, New York,NY, USA; T. M. Fliedner, Ulm, FRG; W. N. Jensen, Washington, DC, USA; S.-A Killmann, Copenhagen, Denmark; L. G. Lajtha, Manchester, England; H. C. Mel, Berkeley, CA, USA; B. Morris, Canberra, Australia; A H. T. Robb-Smith, Oxford, England; S. B. Shohet, San Francisco, CA, USA; B. Thorell, Stockholm, Sweden; 1. G. White, Minneapolis, Minn., USA

Thejoumal is also staffed by an international group of Consulting Editors.

Blood Cells publishes Symposia proceedings that deal with selected topics concerning the Physiology or Pathology of the blood at the cellular level. It also includes original papers and editorials, both invited and unsolicited, submitted by distin­guished international scientists. A unique feature is the publication, following the paper of a commentary by a reviewer and the author's reply.

The emphasis on alterations in structure and function at the cellular level makes this new journal important to every researcher concerned with the Biochemistry, Physiology, Ultra­structure, Immunology and Biophysics of blood cells in health and disease.

Volume 1, Number 1 Unclassifiable Leukemias

Volume I, Number 2 Red Cell Form and Deformability

Volume I, Number 3 Stress Erythropoiesis

Volume 2, Number 1-2 Hemopoietic Dysplasias (Preleukemic States)

Volume 2, Number 3 White Cell Movements

Volume 3, Number I Blood Cell Rheolo?Jl

Volume 3, Number 2 Red Cell Rheolo?Jl (II)

Volume 3, Number 3 Leukemic Cell Surveillance

Volume 4, Number I Diamond Blac/ifan and Cell Differentiation