Anatomia, Histologia, Embryologia
SHORT COMMUNICATION
A Practical Technique for Electron Microscopy of Buffy Coats in Dogs and Cats A. Yabuki*, M. Sawa, H.-S. Chang and O. Yamato Address of authors: Laboratory of Veterinary Clinical Pathology, Joint Faculty of Veterinary Medicine, Kagoshima University, Korimoto, Kagoshima, Japan
*Correspondence: Tel.:/fax: +81 99 285 3561; e-mail: [email protected] With 3 figures Received March 2014; accepted for publication July 2014 doi: 10.1111/ahe.12143
Summary Plastic hematocrit tubes (PHTs) are convenient tools for electron microscopy (EM) of peripheral blood buffy coats, and the PHT-EM technique is expected to be a practical method for veterinary clinical medicine. In this study, fixatives composed of various concentrations of sucrose, glutaraldehyde, and phosphate buffer (PB) were tested for preparing canine and feline buffy coats. The highest quality images were obtained using a fixative consisting of 2.5% glutaraldehyde in 0.1 M PB, and it was concluded that this method allows clinicians who are inexperienced in histological techniques can conveniently transport buffy coat samples to diagnostic laboratories for analysis by EM.
Introduction Electron microscopy (EM) of peripheral blood buffy coats is an important diagnostic tool for various diseases such as leukemia (Dvorak et al., 1981), lysosomal storage diseases (Anderson et al., 2006), and blood infections (Buoro et al., 1989). The standard method of preparing buffy coats for EM uses Wintrobe tubes (Grindem, 1985; Dykstra, 1993) and produces high-quality images of leukocytes and platelets. However, the handling of samples using this method is complicated and requires great care, making it very challenging to transport samples from veterinary clinics to diagnostic laboratories. Moreover, Wintrobe tubes are not used routinely in general veterinary clinics. Plastic hematocrit tubes (PHTs) are convenient implements for the preparation of buffy coats for EM (Hattori, 1970; Mills et al., 1990), allowing very small amounts of blood to be examined. Although the PHT-EM method has long been used in human medicine, it has not been applied practically in veterinary medicine. Centrifuges for hematocrit tubes are commonly found in veterinary clinics; therefore, an EM sample preparation method using PHTs can be a potentially useful tool for the diagnosis of canine and feline diseases. However, to obtain good quality images of hemocytes from canine and feline blood using PHT-EM, methodological studies in practical settings are required. In this study, we prepared peripheral © 2014 Blackwell Verlag GmbH Anat. Histol. Embryol. 44 (2014) 317–320
blood buffy coats from dogs and cats according to a routine diagnostic method used in general veterinary practice and evaluated the quality of images obtained from the PHT-EM method using different fixatives. Materials and Methods The experimental protocols used in this study were approved by the Institutional Animal Care and Use Committee of Kagoshima University, Kagoshima, Japan (No. VM13030). Peripheral blood samples were collected from clinically healthy dogs and cats (n = 3 for both) and were stored in 0.5-ml tubes containing EDTA-2K (Fujifilm, Tokyo, Japan). In accordance with the routine diagnostic method, blood samples were drawn into plain PHTs (Drummond Scientific, Broomall, PA, USA). After centrifugation at 12 000 rpm (15 000 g) for 5 min, the lengths of the PHTs that contained the buffy coats (approximately 2 mm in length) were cut with a razor. These trimmed tubes were immersed in various fixatives. To investigate the effects of sucrose, solutions of 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB) (pH 7.4), containing 0, 2, 4, 8, and 16% sucrose, were prepared. To investigate the effects of glutaraldehyde and PB concentration, 2.5, 3.75, and 5.0% glutaraldehyde solutions were prepared in 0.1 and 0.2 M PB. PHTs were incubated in each fixative for 60 min and then gently mixed on a
317
Electron Microscopy of Buffy Coats
A. Yabuki et al.
Fig. 1. Longitudinal section of a feline buffy coat prepared using the plastic hematocrit tube electron microscopy (PHT-EM) method with Giemsa staining. Scale bars = 400 lm (left panel) and 40 lm (right panels).
shaker overnight at room temperature. After a thorough wash in 0.1 M PB, the samples were further fixed by immersing the PHTs in a solution of 1% osmium tetroxide in 0.1 M PB for 2 h. The samples were dehydrated using a graded ethanol series (50, 70, 80, 90, 95, and 100% ethanol). Buffy coats were ejected from the tubes using a toothpick while submerged in 80% ethanol; they were then transferred to propylene oxide, before being embedded in epoxy resin (Quetol-812; Nisshin EM, Tokyo, Japan) in flat molds. After polymerization, each sample block was attached to a second resin block, formed with a capsule mold, so that longitudinal sections
(a)
(b)
could be obtained. After removing the resin, the thin sections were stained with Giemsa stain (Iwadare et al., 1990). Ultrathin sections cut from the leukocyte layers were then stained with uranyl acetate and lead and were observed using a transmission electron microscope (H-7000KU; Hitachi, Tokyo, Japan). Results and Discussion The PHT-EM method of buffy coat sample preparation produced an ordered arrangement of platelets, lymphocytes/monocytes, and granulocytes in thin sections (Fig. 1).
(c)
Fig. 2. Electron micrographs of leukocytes from a canine buffy coat prepared using the plastic hematocrit tube electron microscopy (PHT-EM) method. The sample was fixed using a solution of 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB). (a) Image of the lymphocyte/monocyte layer. (b) Image of the granulocyte layer. Eo, eosinophil; Ly, lymphocyte; Mo, monocyte; Ne, neutrophil. (c) Image of the platelet layer. Scale bar = 2 lm.
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© 2014 Blackwell Verlag GmbH Anat. Histol. Embryol. 44 (2014) 317–320
A. Yabuki et al.
Fig. 3. Electron micrographs of leukocytes from buffy coats prepared using the plastic hematocrit tube electron microscopy (PHT-EM) method with different fixatives. (a) Canine lymphocyte, 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB). (b) Feline lymphocyte, 2.5% glutaraldehyde in 0.1 M PB. (c) Feline lymphocyte, 2.5% glutaraldehyde in 0.1 M PB with 16% sucrose. (d) Feline neutrophil, 2.5% glutaraldehyde in 0.1 M PB. (e) Feline neutrophil, 2.5% glutaraldehyde in 0.1 M PB with 16% sucrose. (f) Feline neutrophil, 2.5% glutaraldehyde in 0.2 M PB. High-contrast images were obtained using sucrose-free fixatives (a, b, and d), although mitochondrial swelling was observed in feline lymphocytes (b). High osmolality fixatives considerably decreased image contrast (c, e, and f) but also eliminated mitochondrial swelling (c). Scale bar = 1 lm.
Electron Microscopy of Buffy Coats
(a)
(b)
(c)
(d)
(e)
(f)
Solutions containing various concentrations of sucrose (0, 2, 4, 8, and 16%), glutaraldehyde (2.5, 3.75, and 5.0%), and PB (0.1 and 0.2 M) were tested to determine the composition that would produce optimal fixation and image quality. A solution consisting of 2.5% glutaraldehyde in 0.1 M PB without sucrose provided good contrast in images of leukocytes and platelets (Figs 2 and 3a,b), although mitochondrial swelling was observed in feline lymphocytes (Fig. 3b). A fixative containing 16% sucrose reduced mitochondrial swelling; however, the image contrast was concomitantly reduced (Fig. 3c). In particular, neutrophils were adversely affected by the addition of sucrose, and distinguishing between primary and secondary granules became more difficult at higher sucrose concentrations (Fig. 3d,e). This concentration-dependent decline in image quality was already apparent at a 2% sucrose concentration. Increasing the glutaraldehyde concentration had no obvious effect on the image quality of leukocytes and platelets. A higher concentration of PB resulted in images with reduced contrast, an effect that was observed in solutions containing 2.5, 3.75, or 5.0% glutaraldehyde (Fig. 3f). From these experiments, it was determined that a solution of 2.5% glutaraldehyde in 0.1 M PB was the most suitable fixative for analyzing the ultrastructure of canine © 2014 Blackwell Verlag GmbH Anat. Histol. Embryol. 44 (2014) 317–320
and feline peripheral blood buffy coats using the PHTEM method. As the fixative has to permeate the narrow edges of the cut PHT, this fixation method might be slower than the standard method using Wintrobe tubes, which have wider openings (Dykstra, 1993). The osmolality of the fixative often determines the speed of fixation, and sucrose has been used routinely to adjust the osmolality of EM fixatives (Sabatini et al., 1963; Bone and Denton, 1971). Indeed, in this study, mitochondrial swelling in feline lymphocytes was reduced by adding 16% sucrose. A similar improvement was observed when a higher concentration of PB was used. However, this favorable outcome was negated by poor contrast in the images produced, which is a disadvantage associated with high osmolality fixatives. Mitochondrial swelling was observed as an artifact of the PHT-EM method, typically in feline lymphocytes. As lymphocytes occupied a layer closer to the surface than granulocytes (Fig. 1), it is possible that the artifact resulted from factors other than the fixative. In the standard method of buffy coat preparation for EM, blood is centrifuged at a low speed (e.g., 250 g) to prevent the rupture of organelles (Dykstra, 1993). In the present study, PHTs were subjected to high-speed centrifugation
319
Electron Microscopy of Buffy Coats
(15 000 g; 12 000 rpm), in accordance with routine diagnostic methods used in general veterinary practice. The mitochondria of feline lymphocytes may have experienced excessive mechanical stress during this process. Although the limitation of this artifact cannot be disregarded, the PHT-EM method is, nonetheless, a useful addition to the current repertoire of diagnostic tools for canine and feline diseases, as it enables general veterinary clinicians to efficiently process buffy coat samples for ultrastructural analysis by EM. By applying this method, veterinary clinicians who are inexperienced in histological techniques can conveniently transport buffy coat samples to diagnostic laboratories for analysis by EM. Acknowledgements The authors would like to thank Shinichiro Yoneshige (Kagoshima University, Japan) for his assistance with preliminary experiments in this research. Conflict of interest The authors declare that they have no conflict of interest. References Anderson, G. W., V. V. Smith, I. Brooke, M. Malone, and N. J. Sebire, 2006: Diagnosis of neuronal ceroid lipofuscinosis (Batten disease) by electron microscopy in peripheral blood specimens. Ultrastruct. Pathol. 30, 373–378.
320
A. Yabuki et al.
Bone, Q., and E. J. Denton, 1971: The osmotic effects of electron microscope fixatives. J. Cell Biol. 49, 571–581. Buoro, I. B., R. B. Atwell, J. C. Kiptoon, and M. A. Ihiga, 1989: Feline anaemia associated with Ehrlichia-like bodies in three domestic short-haired cats. Vet. Rec. 125, 434–436. Dvorak, A. M., R. A. Monahan, and G. R. Dickersin, 1981: Diagnostic electron microscopy. I. Hematology: differential diagnosis of acute lymphoblastic and acute myeloblastic leukemia. Use of ultrastructural peroxidase cytochemistry and routine electron microscopic technology. Pathol. Annu. 16, 101–137. Dykstra, M. J., 1993: Preparation of buffy coats for TEM. In: A Manual of Applied Techniques for Biological Electron Microscopy. (M. J. Dykstra, ed.). New York: Plenum Press. pp 64–65. Grindem, C. B., 1985: Ultrastructural morphology of leukemic cells from 14 dogs. Vet. Pathol. 22, 456–462. Hattori, A., 1970: A simple micromethod of preparing peripheral leukocytes and platelets for electron microscopy. Arch. Histol. Jpn. 32, 307–313. Iwadare, T., E. Harada, S. Yoshino, and T. Arai, 1990: A solution for removal of resin from epoxy sections. Stain Technol. 65, 205–220. Mills, A. E., M. Emms, and S. G. Licata, 1990: A simple technique for preparation of bone marrow or peripheral blood buffy coat cells for electron microscopy. Ultrastruct. Pathol. 14, 173–176. Sabatini, D. D., K. Bensch, and R. J. Barrnett, 1963: Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol. 17, 19–58.
© 2014 Blackwell Verlag GmbH Anat. Histol. Embryol. 44 (2014) 317–320
SHORT COMMUNICATION
A Practical Technique for Electron Microscopy of Buffy Coats in Dogs and Cats A. Yabuki*, M. Sawa, H.-S. Chang and O. Yamato Address of authors: Laboratory of Veterinary Clinical Pathology, Joint Faculty of Veterinary Medicine, Kagoshima University, Korimoto, Kagoshima, Japan
*Correspondence: Tel.:/fax: +81 99 285 3561; e-mail: [email protected] With 3 figures Received March 2014; accepted for publication July 2014 doi: 10.1111/ahe.12143
Summary Plastic hematocrit tubes (PHTs) are convenient tools for electron microscopy (EM) of peripheral blood buffy coats, and the PHT-EM technique is expected to be a practical method for veterinary clinical medicine. In this study, fixatives composed of various concentrations of sucrose, glutaraldehyde, and phosphate buffer (PB) were tested for preparing canine and feline buffy coats. The highest quality images were obtained using a fixative consisting of 2.5% glutaraldehyde in 0.1 M PB, and it was concluded that this method allows clinicians who are inexperienced in histological techniques can conveniently transport buffy coat samples to diagnostic laboratories for analysis by EM.
Introduction Electron microscopy (EM) of peripheral blood buffy coats is an important diagnostic tool for various diseases such as leukemia (Dvorak et al., 1981), lysosomal storage diseases (Anderson et al., 2006), and blood infections (Buoro et al., 1989). The standard method of preparing buffy coats for EM uses Wintrobe tubes (Grindem, 1985; Dykstra, 1993) and produces high-quality images of leukocytes and platelets. However, the handling of samples using this method is complicated and requires great care, making it very challenging to transport samples from veterinary clinics to diagnostic laboratories. Moreover, Wintrobe tubes are not used routinely in general veterinary clinics. Plastic hematocrit tubes (PHTs) are convenient implements for the preparation of buffy coats for EM (Hattori, 1970; Mills et al., 1990), allowing very small amounts of blood to be examined. Although the PHT-EM method has long been used in human medicine, it has not been applied practically in veterinary medicine. Centrifuges for hematocrit tubes are commonly found in veterinary clinics; therefore, an EM sample preparation method using PHTs can be a potentially useful tool for the diagnosis of canine and feline diseases. However, to obtain good quality images of hemocytes from canine and feline blood using PHT-EM, methodological studies in practical settings are required. In this study, we prepared peripheral © 2014 Blackwell Verlag GmbH Anat. Histol. Embryol. 44 (2014) 317–320
blood buffy coats from dogs and cats according to a routine diagnostic method used in general veterinary practice and evaluated the quality of images obtained from the PHT-EM method using different fixatives. Materials and Methods The experimental protocols used in this study were approved by the Institutional Animal Care and Use Committee of Kagoshima University, Kagoshima, Japan (No. VM13030). Peripheral blood samples were collected from clinically healthy dogs and cats (n = 3 for both) and were stored in 0.5-ml tubes containing EDTA-2K (Fujifilm, Tokyo, Japan). In accordance with the routine diagnostic method, blood samples were drawn into plain PHTs (Drummond Scientific, Broomall, PA, USA). After centrifugation at 12 000 rpm (15 000 g) for 5 min, the lengths of the PHTs that contained the buffy coats (approximately 2 mm in length) were cut with a razor. These trimmed tubes were immersed in various fixatives. To investigate the effects of sucrose, solutions of 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB) (pH 7.4), containing 0, 2, 4, 8, and 16% sucrose, were prepared. To investigate the effects of glutaraldehyde and PB concentration, 2.5, 3.75, and 5.0% glutaraldehyde solutions were prepared in 0.1 and 0.2 M PB. PHTs were incubated in each fixative for 60 min and then gently mixed on a
317
Electron Microscopy of Buffy Coats
A. Yabuki et al.
Fig. 1. Longitudinal section of a feline buffy coat prepared using the plastic hematocrit tube electron microscopy (PHT-EM) method with Giemsa staining. Scale bars = 400 lm (left panel) and 40 lm (right panels).
shaker overnight at room temperature. After a thorough wash in 0.1 M PB, the samples were further fixed by immersing the PHTs in a solution of 1% osmium tetroxide in 0.1 M PB for 2 h. The samples were dehydrated using a graded ethanol series (50, 70, 80, 90, 95, and 100% ethanol). Buffy coats were ejected from the tubes using a toothpick while submerged in 80% ethanol; they were then transferred to propylene oxide, before being embedded in epoxy resin (Quetol-812; Nisshin EM, Tokyo, Japan) in flat molds. After polymerization, each sample block was attached to a second resin block, formed with a capsule mold, so that longitudinal sections
(a)
(b)
could be obtained. After removing the resin, the thin sections were stained with Giemsa stain (Iwadare et al., 1990). Ultrathin sections cut from the leukocyte layers were then stained with uranyl acetate and lead and were observed using a transmission electron microscope (H-7000KU; Hitachi, Tokyo, Japan). Results and Discussion The PHT-EM method of buffy coat sample preparation produced an ordered arrangement of platelets, lymphocytes/monocytes, and granulocytes in thin sections (Fig. 1).
(c)
Fig. 2. Electron micrographs of leukocytes from a canine buffy coat prepared using the plastic hematocrit tube electron microscopy (PHT-EM) method. The sample was fixed using a solution of 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB). (a) Image of the lymphocyte/monocyte layer. (b) Image of the granulocyte layer. Eo, eosinophil; Ly, lymphocyte; Mo, monocyte; Ne, neutrophil. (c) Image of the platelet layer. Scale bar = 2 lm.
318
© 2014 Blackwell Verlag GmbH Anat. Histol. Embryol. 44 (2014) 317–320
A. Yabuki et al.
Fig. 3. Electron micrographs of leukocytes from buffy coats prepared using the plastic hematocrit tube electron microscopy (PHT-EM) method with different fixatives. (a) Canine lymphocyte, 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB). (b) Feline lymphocyte, 2.5% glutaraldehyde in 0.1 M PB. (c) Feline lymphocyte, 2.5% glutaraldehyde in 0.1 M PB with 16% sucrose. (d) Feline neutrophil, 2.5% glutaraldehyde in 0.1 M PB. (e) Feline neutrophil, 2.5% glutaraldehyde in 0.1 M PB with 16% sucrose. (f) Feline neutrophil, 2.5% glutaraldehyde in 0.2 M PB. High-contrast images were obtained using sucrose-free fixatives (a, b, and d), although mitochondrial swelling was observed in feline lymphocytes (b). High osmolality fixatives considerably decreased image contrast (c, e, and f) but also eliminated mitochondrial swelling (c). Scale bar = 1 lm.
Electron Microscopy of Buffy Coats
(a)
(b)
(c)
(d)
(e)
(f)
Solutions containing various concentrations of sucrose (0, 2, 4, 8, and 16%), glutaraldehyde (2.5, 3.75, and 5.0%), and PB (0.1 and 0.2 M) were tested to determine the composition that would produce optimal fixation and image quality. A solution consisting of 2.5% glutaraldehyde in 0.1 M PB without sucrose provided good contrast in images of leukocytes and platelets (Figs 2 and 3a,b), although mitochondrial swelling was observed in feline lymphocytes (Fig. 3b). A fixative containing 16% sucrose reduced mitochondrial swelling; however, the image contrast was concomitantly reduced (Fig. 3c). In particular, neutrophils were adversely affected by the addition of sucrose, and distinguishing between primary and secondary granules became more difficult at higher sucrose concentrations (Fig. 3d,e). This concentration-dependent decline in image quality was already apparent at a 2% sucrose concentration. Increasing the glutaraldehyde concentration had no obvious effect on the image quality of leukocytes and platelets. A higher concentration of PB resulted in images with reduced contrast, an effect that was observed in solutions containing 2.5, 3.75, or 5.0% glutaraldehyde (Fig. 3f). From these experiments, it was determined that a solution of 2.5% glutaraldehyde in 0.1 M PB was the most suitable fixative for analyzing the ultrastructure of canine © 2014 Blackwell Verlag GmbH Anat. Histol. Embryol. 44 (2014) 317–320
and feline peripheral blood buffy coats using the PHTEM method. As the fixative has to permeate the narrow edges of the cut PHT, this fixation method might be slower than the standard method using Wintrobe tubes, which have wider openings (Dykstra, 1993). The osmolality of the fixative often determines the speed of fixation, and sucrose has been used routinely to adjust the osmolality of EM fixatives (Sabatini et al., 1963; Bone and Denton, 1971). Indeed, in this study, mitochondrial swelling in feline lymphocytes was reduced by adding 16% sucrose. A similar improvement was observed when a higher concentration of PB was used. However, this favorable outcome was negated by poor contrast in the images produced, which is a disadvantage associated with high osmolality fixatives. Mitochondrial swelling was observed as an artifact of the PHT-EM method, typically in feline lymphocytes. As lymphocytes occupied a layer closer to the surface than granulocytes (Fig. 1), it is possible that the artifact resulted from factors other than the fixative. In the standard method of buffy coat preparation for EM, blood is centrifuged at a low speed (e.g., 250 g) to prevent the rupture of organelles (Dykstra, 1993). In the present study, PHTs were subjected to high-speed centrifugation
319
Electron Microscopy of Buffy Coats
(15 000 g; 12 000 rpm), in accordance with routine diagnostic methods used in general veterinary practice. The mitochondria of feline lymphocytes may have experienced excessive mechanical stress during this process. Although the limitation of this artifact cannot be disregarded, the PHT-EM method is, nonetheless, a useful addition to the current repertoire of diagnostic tools for canine and feline diseases, as it enables general veterinary clinicians to efficiently process buffy coat samples for ultrastructural analysis by EM. By applying this method, veterinary clinicians who are inexperienced in histological techniques can conveniently transport buffy coat samples to diagnostic laboratories for analysis by EM. Acknowledgements The authors would like to thank Shinichiro Yoneshige (Kagoshima University, Japan) for his assistance with preliminary experiments in this research. Conflict of interest The authors declare that they have no conflict of interest. References Anderson, G. W., V. V. Smith, I. Brooke, M. Malone, and N. J. Sebire, 2006: Diagnosis of neuronal ceroid lipofuscinosis (Batten disease) by electron microscopy in peripheral blood specimens. Ultrastruct. Pathol. 30, 373–378.
320
A. Yabuki et al.
Bone, Q., and E. J. Denton, 1971: The osmotic effects of electron microscope fixatives. J. Cell Biol. 49, 571–581. Buoro, I. B., R. B. Atwell, J. C. Kiptoon, and M. A. Ihiga, 1989: Feline anaemia associated with Ehrlichia-like bodies in three domestic short-haired cats. Vet. Rec. 125, 434–436. Dvorak, A. M., R. A. Monahan, and G. R. Dickersin, 1981: Diagnostic electron microscopy. I. Hematology: differential diagnosis of acute lymphoblastic and acute myeloblastic leukemia. Use of ultrastructural peroxidase cytochemistry and routine electron microscopic technology. Pathol. Annu. 16, 101–137. Dykstra, M. J., 1993: Preparation of buffy coats for TEM. In: A Manual of Applied Techniques for Biological Electron Microscopy. (M. J. Dykstra, ed.). New York: Plenum Press. pp 64–65. Grindem, C. B., 1985: Ultrastructural morphology of leukemic cells from 14 dogs. Vet. Pathol. 22, 456–462. Hattori, A., 1970: A simple micromethod of preparing peripheral leukocytes and platelets for electron microscopy. Arch. Histol. Jpn. 32, 307–313. Iwadare, T., E. Harada, S. Yoshino, and T. Arai, 1990: A solution for removal of resin from epoxy sections. Stain Technol. 65, 205–220. Mills, A. E., M. Emms, and S. G. Licata, 1990: A simple technique for preparation of bone marrow or peripheral blood buffy coat cells for electron microscopy. Ultrastruct. Pathol. 14, 173–176. Sabatini, D. D., K. Bensch, and R. J. Barrnett, 1963: Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol. 17, 19–58.
© 2014 Blackwell Verlag GmbH Anat. Histol. Embryol. 44 (2014) 317–320
