British Jotrmal of Haernatology, 1975,30, 303.
Surface Features of Human Eosinophils: a Scanning and Transmission Electron Microscopic Study
of a Case of Eosinophilia AARON POLLIACK AND STEVEN D. DOUGLAS*
Department of Hematology, Hadassah University Hospital and Hebrew University-Hadassah Medical School, Jerusalem, Israel, and *Section of Immunology, Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota (Received 27 November 1974; acceptedfor publication 7January 1975) SUMMARY.Cells were obtained from the peripheral blood of a patient with marked non-leukaemic eosinophilia. Transmission electron microscopy (TEM) showed typical mature eosinophils. The surface architecture of eosinophils by scanning electron microscopy (SEM) shows that most were spherical with varying numbers of microvilli; a smaller proportion of cells had ridge-like profiles, small ruffles and occasionally blebs. The surface features of the eosinophil thus resemble most lymphocytes and this finding must be considered when leucocyte populations are examined by SEM. Critical point dried human leucocytes have recently been studied by scanning electron microscopy (SEM) and their surface architecture has been described (Polliack et al, 1973a, b; Wetzel et al, 1973; Lin et al, 1973). Lymphocytes display varying numbers of finger-like microvilli, whereas monocytes have characteristic ruffled membranes and ridge-like profiles (Polliack et al, 1973b, 1g74a, b, 1975 ;Lin et al, 1973). Granulocytes show a diversity of surface structures (Wetzel et al, 1g73), but frequently show ridge profiles and ruffles. Because of this overlap of surface features, the distinction between granulocytes and monocytes in mixed leucocyte populations is difficult on the basis ofsurface architecture alone (Wetzel et al, 1973). The surface morphology of eosinophils and basophils has not been clearly defined, primarily due to difficulties in obtaining concentrated samples of these cells attributable to their small numbers in the peripheral circulation and the relative rarity of pathologic conditions involving these cell types. Nevertheless, the limited available data suggests that they too may display diverse surface features (Wetzel et al, 1973). W e have had the opportunity to study by TEM and SEM, cells from an unusual patient with large numbers of circulating eosinophils, nonleukaemic, but associated with malignant lymphoma.
Case Report A 61-year-old black man was admitted for the investigation of generalized lymphadenopathy, pruritus and eosinophilia of I year’s duration. There was no history of fever or weight Correspondence: Dr Steven D. Douglas, Department of Medicine, University of Minnesota Medical School, Mayo Building, Minneapolis, Minnesota 55455, U.S.A.
303
304
Aaron Polliack and Steven D. Douglas
loss. Physical examination revealed generalized lymphadenopathy, mild hepatomegaly and no splenomegaly. The blood count showed a haemoglobin of 14.3 g/dl, haematocrit of 0.41, platelets 240 x 10’11. The white blood cell count was 4.8-6.5 x 10~11with 85-95% mature eosinophils. Examination of the bone marrow revealed that 70-80% of the cells of the myeloid series were mature eosinophils. Chromosomal karyotypes, leucocyte alkaline phosphatase and eosinophil function studies including phagocytosis, oxygen consumption and glucose oxidation were within normal limits. Serum vitamin B,, was 602 ng/l, LDH was 1650 with increase of isoenzyme fractions I and 2, and the alkaline phosphatase was 140 units. Chest roentgenograms, skeletal survey and liver scan were normal. A lymph node biopsy showed a malignant lymphoma of the poorly differentiated type. The lymphadenopathy increased and the white cell count rose to 96 x 10’11 (86-98% eosinophils). 40 mg prednisone per day was administered and the total leucocyte count fell dramatically within a week to 7.0 x 10~11 with 5% eosinophils. The lymphadenopathy disappeared and the patient was discharged on 20 mg prednisone daily. One month later the WBC was 6.9 x 10’11 with 64% neutrophils, 1% monocytes, 4% eosinophils and 31% lymphocytes. A bone marrow aspirate was within normal limits with 6% eosinophils and 7% mast cells. MATERIALS AND METHODS Transmission Electron Microscopy (TEM).Pellets were fixed with cacodylate buffered 1.5% glutaraldehyde (pH 7.4, 4”C), postfixed in 1% buffered osmium tetroxide, dehydrated, embedded in Epon and sectioned with an LKB Ultrotome 111. Thin sections were stained with uranyl acetate and lead citrate, and examined with a Siemens 102 electron microscope operated at 80 kV. Scanning Electron Microscopy (SEM). A buffy coat was obtained by centrifugation of heparinized whole blood at 1000rpm for 8 min. Cells were resuspended in Hanks’ Balanced Salt Solution (HBSS) (pH 7.3, 310-320 mosmol). Differential counts were done on WrightGiemsa stained smears of the same samples. 6 x 106 cells were collected in monolayer-like distribution on silver membranes by aspiration as described in earlier studies (Polliacket al,1g73a, by1974a, c, 1975). The cells were fixed, dehydrated, placed in amyl acetate or Freon 113 and critical point dried in Freon 13 (Cohen et al, 1968) or carbon dioxide (Anderson, 1951). A Cambridge S4 scanning rnicroscope with IOO or ZOO ,um diameter illuminating aperatures were used at an accelerating voltage of 10-20 kV. Aicrographs were recorded on Polaroid type 55 P / N film at direct magnification from x 1000 to 14 000. RESULTS Ninety-eight per cent of the cells examined by light microscopy were mature eosinophilic leucocytes which appeared normal. Examination of thin sections revealed typical mature eosinophils (Zucker-Franklin, 1968). The nuclei were predominantly heterochromatic and the cytoplasm contained a small Golgi zone, mitochondria, ribosomes and characteristic crystalloid containing granules (Fig I). Cells with prominent rough surfaced endoplasmic reticulum or large inclusion granules, an index of immaturity (Zucker-Franklin, 1g74), were
Surface Features of H m a i z Eosiiiophils
FIG I . TEM. Low power micrographs showing portions of six cosiiiophils. Granules containing typical crystalloids arc cvident. x 7500. (FQCi%'p 304)
Aarori Polliack and Steveiz D. Douglas
Szrrface Features of H u m a n Eosinophils
30.5
not obscrved. The small cytoplasmic granule described by Parmley & Spicer (1974) requires cytochemical study and was not identified. As in previous studies (Polliack et al, 1973b, 1974a, b, c, 1975; Lin et al, 1973) the cells examined by SEM were classified on the basis of their surface architecture.Cells with relatively smooth surfaces and few microvilli were scored as ‘smooth‘ (Figs z and 3 ) ; those with moderately and markedly villous surfaces were designated as ‘villous’(Figs 4 and 5 ) and were distinguished from cells displaying mainly ridge-like profiles and ruffled membranes (Figs 6 and 7). Ridge-like profiles were seen as narrow raised transverse or longitudinal surface folds (100-200mp thick; generally 0.6-2.0 p in length) (Figs 6 8 ) . Ruffled membranes are defined as broader based undulating cytoplasmic veils frequently very well developed and often concentrated at the cell margin (generally 100-300 mp broad), varying in length usually up to 1.5-3.5 p (Figs 8 and 9). A differential count was performed on 140 cells by SEM. Fifty-two per cent of the cells were of the smooth type; 24% were villous, 18% had ridge-like profiles; 4% displayed a small number of moderately well developed ruffles in addition to ridge-like profiles; and 2% had both microvilli and surface blebs.
DISCUSSION This is a rare example of a patient with eosinophilia in response to malignancy. By light and transmission electron inicroscopy the eosinophils from this patient were mature and indistinguishable from normals. By SEM the eosinophils showed a spectrum of surface morphology, most having either smooth or villous surfaces while others displayed ridge-like profiles and small ruffles which are generally seen in the majority of neutrophils. Recently, the SEM features of critical point dried human leucocytes have been described (Polliack et al, 1973a, b, 1974a, by c; Wetzel et al, 1973 ; Lin et al, 1973). Lymphocytes are spherical with varying numbers of microvilli, while monocytes characteristicallyhave ruffled membranes and ridgelike profiles with a relatively small number of microvilli (Polliack et al, 1973b, 1974a, b, 1975 ; Lin et a/, 1973). The leukaemic counterparts of these cells generally have the same surface characteristics (Polliack et al, 1g74a, b, 1975). Granulocytes have a diverse surface morphology (Wetzel et al, 1973) but most of them, presumably neutrophils, have ridge-like profiles. Until now the surface architecture of normal basophils and eosinophils has not been adequately defined by SEM because of their insufficient numbers in the peripheral circulation, difficulties in obtaining concentrated samples, and the rarity of pathologic states iiivolving these cell types. Leukaemic rat basophils have recently been shown (Braylan, 1974) to have a spectrum of surface morphology similar to the eosinophils described in the present report; it is possible that normal human basophils FIG2. SEM. Three eosinophils showing smooth and villous surfaces. Note thc bottom right cell has small ruffled membranes. x 6300. FIG 3 . SEM. Eosinophil with smooth surface with small number of stub-like microvilli. x 9100. FIG 4. Eosinophil with moderate number of rnicrovilli. x 9100. FIG5 . Eosinophil with villous surface and large number of microvilli. x 9100. FIG6 . Eosinophil showing raised ridge-like profdes and some short microvilli. x 9100. FIG 7. Eosinophil with well-developed ridge-like profiles. x 8400. D
FIG8. Group of eosiiiophils showing s m o o t h and villous surfaccs. T w o cclls show ridge-likc profilcs and s m a l l short ruffles. x 9100. FIG y. Group of eosiriophils, three of which arc s m o o t h , wliilc thc othcr t w o \Iio\v iiiicroviili, r u t h , and ridge-like profilcs. x gXoo.
306
Aaron Polliack and Steven D. Douglas
have similar surface structures. Thus, when mixed populations of leucocytes are examined, it must be realized that eosinophils and basophils show varying numbers of microvilli and could be misidentified as lymphocytes. Using a positive method for identification of cells by light microscopy and SEM, Wetzel et al (1973)have indicated this problem. The small numbers of eosinophils and basophils present in the peripheral blood, however, and their even smaller representation in mononuclear cell suspensions prepared by Ficoll-Hypaque gradient centrifugation(Boyum, 1968), would tend to minimize the error for normal samples examined by SEM. ACKNOWLEDGMENTS
This work was supported by grants from the Leukemia Research Foundation, Pelham Parkway League for Grants for Cancer Research (A. Polliack) and by the National Institutes of Health, A1 12478-01 and the National Leukemia Association (S. D. Douglas). W e acknowledge the technical assistance of Ms Elizabeth Chen and Ms Mary Ooka. REFERENCES ANDERSON, T.F. (1951) Techniques for preservation of three-dimcnsional structure in preparing specimens for the electron microscope. Transactions ofthe New York Academy of Sciences, 13, 130. B ~ Y U MA., (1968) Separation of leukocytes from blood and bone marrow. Scandinavian Journal qf Clinical and Laboratory Investigation, Supplement, 218,97. BRAYLAN, R. (1974) Scanning electron niicroscopy of rat basophilic leukemia cells. (Submitted for publication.) COHEN,A.L., MARLOW, D.P. & GARNER, G.E. (1968) A rapid critical point method using fluorocarbons (‘freons’) as intermediate and transitional fluids. Journal de Microscopie, 7, 331. LIN, P.S., COOPER,A.G. & WORTIS,H.H. (1973) Scanning electron microscopy of human T cell and B cell rosettes. New England Journal of Medicine, 289%548. PARMLEY, R.T. & SPICER,S.S. (1974) Cytochemical and ultrastructural identification of a small type granule in human late eosinophils. Laboratory Investigation, 30, 557. POLLIACK, A., LAMPEN, N. & DE HARVEN,E. (1g73a) Comparison of air drying and critical point drying procedures for the study of human blood cells by scanning electron microscopy. Proceedings of 6th Annual Scanning Electron Microscopy Symposium, I I T R I , Chicago, Illinois, U.S.A., p 535. POLLIACK,A., LAMPEN,N., CLARKSON, B.D., DE HARVEN, E., BENTWICH, Z., SIEGAL, F.P. & KUNKEL, H.G. (1g73b) Identification of human B and T
lyrnphocytcs by scanning electron microscopy. Journal of Experimental Medicine, 138, 607. POLLIACK, A., LAMPEN, N. & DE HAKVEN, E. (1974a) Scanning electron microscopy of lymphocytes of known B and T derivation. Proceedings of 7th Annual Scanning Electron Microscopy Symposiuni, IITRI, Chicago, Illinois, U.S.A., p 673. POLLIACK, A., Fu, S.M., DOUGLAS, S.D., BENTWICH, Z. 61 DE HARVEN, E. (1974b) Scanning electron microscopy of human lymphocyte-sheep erythrocyte rosettes. Journal ofExperimenta1 Medicine, 140, 146. POLLIACK, A., SIFJGAL, F.P., Fu, S.M., CLARKSON, B.D., WINCHESTER, R.J., LAMPEN,N., SIEGAL,M. & DE HARVEN, E. (1974~) Surface morphology of leukemic lymphocytes: An SEM study of 84 cases of acute and chronic lymphocytic leukemias and related lymphoproliferative disorders. (Submitted for publication.) POLLIACK,A. & DE HARVEN,E. (1975) Surface features of normal and leukemic lymphocytes as seen by scanning electron microscopy. Clirzical Imniunology and Immunopathology, 3, 412. WETZEL,B., ERICKSON,B.W., JR & LEVIS, W.R. (1973) The need for positive identification of leukocytes examined by SEM. Proceedings of 6th Annual Scanning Electron Microscopy Symposium, IITRI, Chicago, Illinois, U S A . , p 535. ZUCKER-FRANKLIN, D. (1968) Electron microscopic studies of human granulocytes: structural variations related to function. Seminars in Hematology, 5, 19. ZUCKER-FRANKLIN, D. (1974) Eosinophil function and disorders. Advances in Internal Medicine, 19, I.
Surface Features of Human Eosinophils: a Scanning and Transmission Electron Microscopic Study
of a Case of Eosinophilia AARON POLLIACK AND STEVEN D. DOUGLAS*
Department of Hematology, Hadassah University Hospital and Hebrew University-Hadassah Medical School, Jerusalem, Israel, and *Section of Immunology, Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota (Received 27 November 1974; acceptedfor publication 7January 1975) SUMMARY.Cells were obtained from the peripheral blood of a patient with marked non-leukaemic eosinophilia. Transmission electron microscopy (TEM) showed typical mature eosinophils. The surface architecture of eosinophils by scanning electron microscopy (SEM) shows that most were spherical with varying numbers of microvilli; a smaller proportion of cells had ridge-like profiles, small ruffles and occasionally blebs. The surface features of the eosinophil thus resemble most lymphocytes and this finding must be considered when leucocyte populations are examined by SEM. Critical point dried human leucocytes have recently been studied by scanning electron microscopy (SEM) and their surface architecture has been described (Polliack et al, 1973a, b; Wetzel et al, 1973; Lin et al, 1973). Lymphocytes display varying numbers of finger-like microvilli, whereas monocytes have characteristic ruffled membranes and ridge-like profiles (Polliack et al, 1973b, 1g74a, b, 1975 ;Lin et al, 1973). Granulocytes show a diversity of surface structures (Wetzel et al, 1g73), but frequently show ridge profiles and ruffles. Because of this overlap of surface features, the distinction between granulocytes and monocytes in mixed leucocyte populations is difficult on the basis ofsurface architecture alone (Wetzel et al, 1973). The surface morphology of eosinophils and basophils has not been clearly defined, primarily due to difficulties in obtaining concentrated samples of these cells attributable to their small numbers in the peripheral circulation and the relative rarity of pathologic conditions involving these cell types. Nevertheless, the limited available data suggests that they too may display diverse surface features (Wetzel et al, 1973). W e have had the opportunity to study by TEM and SEM, cells from an unusual patient with large numbers of circulating eosinophils, nonleukaemic, but associated with malignant lymphoma.
Case Report A 61-year-old black man was admitted for the investigation of generalized lymphadenopathy, pruritus and eosinophilia of I year’s duration. There was no history of fever or weight Correspondence: Dr Steven D. Douglas, Department of Medicine, University of Minnesota Medical School, Mayo Building, Minneapolis, Minnesota 55455, U.S.A.
303
304
Aaron Polliack and Steven D. Douglas
loss. Physical examination revealed generalized lymphadenopathy, mild hepatomegaly and no splenomegaly. The blood count showed a haemoglobin of 14.3 g/dl, haematocrit of 0.41, platelets 240 x 10’11. The white blood cell count was 4.8-6.5 x 10~11with 85-95% mature eosinophils. Examination of the bone marrow revealed that 70-80% of the cells of the myeloid series were mature eosinophils. Chromosomal karyotypes, leucocyte alkaline phosphatase and eosinophil function studies including phagocytosis, oxygen consumption and glucose oxidation were within normal limits. Serum vitamin B,, was 602 ng/l, LDH was 1650 with increase of isoenzyme fractions I and 2, and the alkaline phosphatase was 140 units. Chest roentgenograms, skeletal survey and liver scan were normal. A lymph node biopsy showed a malignant lymphoma of the poorly differentiated type. The lymphadenopathy increased and the white cell count rose to 96 x 10’11 (86-98% eosinophils). 40 mg prednisone per day was administered and the total leucocyte count fell dramatically within a week to 7.0 x 10~11 with 5% eosinophils. The lymphadenopathy disappeared and the patient was discharged on 20 mg prednisone daily. One month later the WBC was 6.9 x 10’11 with 64% neutrophils, 1% monocytes, 4% eosinophils and 31% lymphocytes. A bone marrow aspirate was within normal limits with 6% eosinophils and 7% mast cells. MATERIALS AND METHODS Transmission Electron Microscopy (TEM).Pellets were fixed with cacodylate buffered 1.5% glutaraldehyde (pH 7.4, 4”C), postfixed in 1% buffered osmium tetroxide, dehydrated, embedded in Epon and sectioned with an LKB Ultrotome 111. Thin sections were stained with uranyl acetate and lead citrate, and examined with a Siemens 102 electron microscope operated at 80 kV. Scanning Electron Microscopy (SEM). A buffy coat was obtained by centrifugation of heparinized whole blood at 1000rpm for 8 min. Cells were resuspended in Hanks’ Balanced Salt Solution (HBSS) (pH 7.3, 310-320 mosmol). Differential counts were done on WrightGiemsa stained smears of the same samples. 6 x 106 cells were collected in monolayer-like distribution on silver membranes by aspiration as described in earlier studies (Polliacket al,1g73a, by1974a, c, 1975). The cells were fixed, dehydrated, placed in amyl acetate or Freon 113 and critical point dried in Freon 13 (Cohen et al, 1968) or carbon dioxide (Anderson, 1951). A Cambridge S4 scanning rnicroscope with IOO or ZOO ,um diameter illuminating aperatures were used at an accelerating voltage of 10-20 kV. Aicrographs were recorded on Polaroid type 55 P / N film at direct magnification from x 1000 to 14 000. RESULTS Ninety-eight per cent of the cells examined by light microscopy were mature eosinophilic leucocytes which appeared normal. Examination of thin sections revealed typical mature eosinophils (Zucker-Franklin, 1968). The nuclei were predominantly heterochromatic and the cytoplasm contained a small Golgi zone, mitochondria, ribosomes and characteristic crystalloid containing granules (Fig I). Cells with prominent rough surfaced endoplasmic reticulum or large inclusion granules, an index of immaturity (Zucker-Franklin, 1g74), were
Surface Features of H m a i z Eosiiiophils
FIG I . TEM. Low power micrographs showing portions of six cosiiiophils. Granules containing typical crystalloids arc cvident. x 7500. (FQCi%'p 304)
Aarori Polliack and Steveiz D. Douglas
Szrrface Features of H u m a n Eosinophils
30.5
not obscrved. The small cytoplasmic granule described by Parmley & Spicer (1974) requires cytochemical study and was not identified. As in previous studies (Polliack et al, 1973b, 1974a, b, c, 1975; Lin et al, 1973) the cells examined by SEM were classified on the basis of their surface architecture.Cells with relatively smooth surfaces and few microvilli were scored as ‘smooth‘ (Figs z and 3 ) ; those with moderately and markedly villous surfaces were designated as ‘villous’(Figs 4 and 5 ) and were distinguished from cells displaying mainly ridge-like profiles and ruffled membranes (Figs 6 and 7). Ridge-like profiles were seen as narrow raised transverse or longitudinal surface folds (100-200mp thick; generally 0.6-2.0 p in length) (Figs 6 8 ) . Ruffled membranes are defined as broader based undulating cytoplasmic veils frequently very well developed and often concentrated at the cell margin (generally 100-300 mp broad), varying in length usually up to 1.5-3.5 p (Figs 8 and 9). A differential count was performed on 140 cells by SEM. Fifty-two per cent of the cells were of the smooth type; 24% were villous, 18% had ridge-like profiles; 4% displayed a small number of moderately well developed ruffles in addition to ridge-like profiles; and 2% had both microvilli and surface blebs.
DISCUSSION This is a rare example of a patient with eosinophilia in response to malignancy. By light and transmission electron inicroscopy the eosinophils from this patient were mature and indistinguishable from normals. By SEM the eosinophils showed a spectrum of surface morphology, most having either smooth or villous surfaces while others displayed ridge-like profiles and small ruffles which are generally seen in the majority of neutrophils. Recently, the SEM features of critical point dried human leucocytes have been described (Polliack et al, 1973a, b, 1974a, by c; Wetzel et al, 1973 ; Lin et al, 1973). Lymphocytes are spherical with varying numbers of microvilli, while monocytes characteristicallyhave ruffled membranes and ridgelike profiles with a relatively small number of microvilli (Polliack et al, 1973b, 1974a, b, 1975 ; Lin et a/, 1973). The leukaemic counterparts of these cells generally have the same surface characteristics (Polliack et al, 1g74a, b, 1975). Granulocytes have a diverse surface morphology (Wetzel et al, 1973) but most of them, presumably neutrophils, have ridge-like profiles. Until now the surface architecture of normal basophils and eosinophils has not been adequately defined by SEM because of their insufficient numbers in the peripheral circulation, difficulties in obtaining concentrated samples, and the rarity of pathologic states iiivolving these cell types. Leukaemic rat basophils have recently been shown (Braylan, 1974) to have a spectrum of surface morphology similar to the eosinophils described in the present report; it is possible that normal human basophils FIG2. SEM. Three eosinophils showing smooth and villous surfaces. Note thc bottom right cell has small ruffled membranes. x 6300. FIG 3 . SEM. Eosinophil with smooth surface with small number of stub-like microvilli. x 9100. FIG 4. Eosinophil with moderate number of rnicrovilli. x 9100. FIG5 . Eosinophil with villous surface and large number of microvilli. x 9100. FIG6 . Eosinophil showing raised ridge-like profdes and some short microvilli. x 9100. FIG 7. Eosinophil with well-developed ridge-like profiles. x 8400. D
FIG8. Group of eosiiiophils showing s m o o t h and villous surfaccs. T w o cclls show ridge-likc profilcs and s m a l l short ruffles. x 9100. FIG y. Group of eosiriophils, three of which arc s m o o t h , wliilc thc othcr t w o \Iio\v iiiicroviili, r u t h , and ridge-like profilcs. x gXoo.
306
Aaron Polliack and Steven D. Douglas
have similar surface structures. Thus, when mixed populations of leucocytes are examined, it must be realized that eosinophils and basophils show varying numbers of microvilli and could be misidentified as lymphocytes. Using a positive method for identification of cells by light microscopy and SEM, Wetzel et al (1973)have indicated this problem. The small numbers of eosinophils and basophils present in the peripheral blood, however, and their even smaller representation in mononuclear cell suspensions prepared by Ficoll-Hypaque gradient centrifugation(Boyum, 1968), would tend to minimize the error for normal samples examined by SEM. ACKNOWLEDGMENTS
This work was supported by grants from the Leukemia Research Foundation, Pelham Parkway League for Grants for Cancer Research (A. Polliack) and by the National Institutes of Health, A1 12478-01 and the National Leukemia Association (S. D. Douglas). W e acknowledge the technical assistance of Ms Elizabeth Chen and Ms Mary Ooka. REFERENCES ANDERSON, T.F. (1951) Techniques for preservation of three-dimcnsional structure in preparing specimens for the electron microscope. Transactions ofthe New York Academy of Sciences, 13, 130. B ~ Y U MA., (1968) Separation of leukocytes from blood and bone marrow. Scandinavian Journal qf Clinical and Laboratory Investigation, Supplement, 218,97. BRAYLAN, R. (1974) Scanning electron niicroscopy of rat basophilic leukemia cells. (Submitted for publication.) COHEN,A.L., MARLOW, D.P. & GARNER, G.E. (1968) A rapid critical point method using fluorocarbons (‘freons’) as intermediate and transitional fluids. Journal de Microscopie, 7, 331. LIN, P.S., COOPER,A.G. & WORTIS,H.H. (1973) Scanning electron microscopy of human T cell and B cell rosettes. New England Journal of Medicine, 289%548. PARMLEY, R.T. & SPICER,S.S. (1974) Cytochemical and ultrastructural identification of a small type granule in human late eosinophils. Laboratory Investigation, 30, 557. POLLIACK, A., LAMPEN, N. & DE HARVEN,E. (1g73a) Comparison of air drying and critical point drying procedures for the study of human blood cells by scanning electron microscopy. Proceedings of 6th Annual Scanning Electron Microscopy Symposium, I I T R I , Chicago, Illinois, U.S.A., p 535. POLLIACK,A., LAMPEN,N., CLARKSON, B.D., DE HARVEN, E., BENTWICH, Z., SIEGAL, F.P. & KUNKEL, H.G. (1g73b) Identification of human B and T
lyrnphocytcs by scanning electron microscopy. Journal of Experimental Medicine, 138, 607. POLLIACK, A., LAMPEN, N. & DE HAKVEN, E. (1974a) Scanning electron microscopy of lymphocytes of known B and T derivation. Proceedings of 7th Annual Scanning Electron Microscopy Symposiuni, IITRI, Chicago, Illinois, U.S.A., p 673. POLLIACK, A., Fu, S.M., DOUGLAS, S.D., BENTWICH, Z. 61 DE HARVEN, E. (1974b) Scanning electron microscopy of human lymphocyte-sheep erythrocyte rosettes. Journal ofExperimenta1 Medicine, 140, 146. POLLIACK, A., SIFJGAL, F.P., Fu, S.M., CLARKSON, B.D., WINCHESTER, R.J., LAMPEN,N., SIEGAL,M. & DE HARVEN, E. (1974~) Surface morphology of leukemic lymphocytes: An SEM study of 84 cases of acute and chronic lymphocytic leukemias and related lymphoproliferative disorders. (Submitted for publication.) POLLIACK,A. & DE HARVEN,E. (1975) Surface features of normal and leukemic lymphocytes as seen by scanning electron microscopy. Clirzical Imniunology and Immunopathology, 3, 412. WETZEL,B., ERICKSON,B.W., JR & LEVIS, W.R. (1973) The need for positive identification of leukocytes examined by SEM. Proceedings of 6th Annual Scanning Electron Microscopy Symposium, IITRI, Chicago, Illinois, U S A . , p 535. ZUCKER-FRANKLIN, D. (1968) Electron microscopic studies of human granulocytes: structural variations related to function. Seminars in Hematology, 5, 19. ZUCKER-FRANKLIN, D. (1974) Eosinophil function and disorders. Advances in Internal Medicine, 19, I.
