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Cytochemical identification of lymphocytes and other mononuclear cells in ovine and bovine hemal nodes
Comp. lmmun. Microbiol. Offbct. Dis. Vol. 9, No. 4, pp. 297 302, 1986 Printed in Great Britain
0147-9571/86 $3.00+0.00 Pergamon Journals Ltd
C Y T O C H E M I C A L I D E N T I F I C A T I O N OF L Y M P H O C Y T E S A N D O T H E R M O N O N U C L E A R CELLS IN O V I N E AND BOVINE HEMAL NODES P. CECCARELLI, A. M. GARGIULO, O. FAGIOLI a n d V. PEDINI Istituto di Anatomia Normale Veterinaria, Via S. Costanzo, 4 06100 Perugia, Italy Abstraet~Cytochemical and immunocytochemical studies were carried out with specific markers for B lymphocytes, dendritic reticulum cells (ATPase, 5'Nase, SIg) and T lymphocytes (ANAE, A.P.) in an attempt to identify the mononuclear cells present in bovine and ovine hemal nodes. The results show that primary nodes and mantle of secundary nodes are composed of B cells, whereas T cells are mainly localized in the interfollicular cords. Since such an arrangement resembles the picture in normal lymph nodes, but the direct contact between lymphatic tissue and blood is more reminiscent of the spleen, hemal nodes probably perform immunological functions similar to those of both normal lymph nodes and spleen.
Key words: Hemal node, ruminants, cytochemistry, immunocytochemistry, B lymphocyte, T lymphocyte
R~sum&-On a cherch6 ~iidentifier les cellules mononucl~aires pr~sentes dans les noeuds h+matiques (H.N.) des ovins et des bovins avec des m+thodes d'histochimie et d'himmunohistochimie. On a mis en 6vidence des lymphocytes B, des cellules r+ticulaires dendritiques et des lymphocytes T, en employant des marquers 6nzymatiques sp6cifiques: pour les lymphocytes B e t les cellules r&iculaires, l'ATPase, la 5'Nase et les immunoglobulines de surface; et pour les lymphocytes T, I'ANAE et I'A.P. Les lymphocytes B constituent les nodules primaires et la portion p+riph6rique des nodules secondaires; les lymphocytes T sont accumul~s surtout dans les cordons interfolliculaires. Cette disposition du tissue lymphatique ressemble h celle d'un classique noeud lymphatique mais, d'autre part, le contact direct entre le tissu lymphatique et le sang rappelle la structure de la rate. On peut ainsi conclure que les noeuds h6matiques jouent le m6me r61e immunologique que les noeuds lymphatiques et la rate.
Mots-clefs: Noeud h6matique, ruminants, histochimie, immunohistochimie, lymphocyte B, lymphocyte T
INTRODUCTION T h e f u n c t i o n o f h e m a l n o d e s , i.e. the l y m p h a t i c o r g a n s o f r u m i n a n t s r e l a t e d to the b l o o d b u t n o t to the l y m p h c i r c u l a t i o n , is still i n c o m p l e t e l y u n d e r s t o o d . A c c o r d i n g to e a r l y r e p o r t s , these h e m o l y m p h a t i c o r g a n s m i g h t be i n v o l v e d w i t h e i t h e r the e r y t h r o p o i e s i s [!] o r the m y e l o p o i e s i s [1,2]. H o w e v e r , m o r e r e c e n t l y it has b e e n s u g g e s t e d t h a t h e m a l n o d e s m a y p l a y a role in the i m m u n e r e s p o n s e [3-5]. I n p r e v i o u s studies it was s h o w n t h a t the r a t e o f B a n d T l y m p h o c y t e s in b o v i n e h e m a l n o d e s is s i m i l a r to t h a t f o u n d in the p e r i p h e r a l b l o o d [5]. T h e p r e s e n t p a p e r r e p o r t s a c y t o c h e m i c a l s t u d y w h e r e e n z y m e m a r k e r s specific for l y m p h o i d a n d m o n o c y t i c cells w e r e used in o r d e r to i d e n t i f y a n d to l o c a l i z e the cells in the h e m a l n o d e s o f r u m i n a n t s . 297 (IMID
94
A
298
P. CECCARELLI et al.
MATERIALS AND METHODS Hemal nodes were removed from 15 healthy cattle and 15 healthy sheep of both sexes, shortly after slaughter. Small fragments of each hemal node were frozen by immersion in iso-pentane pre-cooled with liquid nitrogen and then sectioned at 8 ~m in a cryostat. Cells making the parenchyma of the H.N. were identified by demonstrating the following enzymatic activities: ATPase, 5' nucleotidase (5' Nase), non-specific acid-c~-naphthylacetate esterase (ANAE) and acid phosphatase (A.P.) For ATPase and 5'Nase activity demonstration, cryostat sections were fixed for 15-30 min with 4% fresh formalin buffered with 0.1 M Na-cacodylate pH 7.2 and then incubated in a medium prepared according to Wachstein and Meisel [6]. The ANAE activity was revealed on fixed sections incubated in 50 ml of 0.1 M sodium acetate pH 5.2 containing 20 mg cc-naphthyl acetate and 15 mg fast garnet GBC [7]. The A.P. activity was revealed incubating the fixed sections in 10 ml 0.1 M acetate buffer pH 5.0 containing 2mg naphthol AS-BI phosphoric acid (dissolved in 0.1 ml N,N-dimethyl-formamide) and 10 mg fast garnet GBC. All reagents, except sodium acetate and acetic acid, were from Sigma Chemical Co. Controls were performed incubating the fixed sections in a medium without substrate. In the sheep hemal nodes the cells bearing surface immunoglobulins (SIg) have been also identified using an indirect immunoperoxidase technique according to Sternberger [8]. An anti-sheep IgG serum raised in rabbit (Miles-Yeda Ltd) has been used as first antibody. Sections incubated in a medium where first specific antibody was substituted with PBS served as controls.
RESULTS Morphological studies revealed both in bovine and ovine two types of nodules within the hemal nodes: "primary nodules" containing exclusively the small lymphocytes, and "secondary nodules" consisting of a light staining germinal center composed of large lymphoid cells and macrophages, surrounded by a darker mantle of small lymphocytes (Fig. 1). The small lymphocytes of the primary nodules and those making the wall of the secondary nodules were intensely ATPase (Fig. 2) and 5'Nase (Fig. 3) positive. Dendritic reticular cells and the endothelial-like cells that lined the blood sinus walls also displayed 5'Nase activity. Immunohistochemical studies showed that almost all the cells of the primary nodule and the mantle cells of secondary nodule, expressed SIg (Figs 4 and 5). When the mononuclear cells were subjected to the ANAE reaction, they showed two distinct staining patterns: i.e. of granular or diffuse type, respectively. According to this finding two types of cells seem to be involved. Both were localized primarily in the cords of lymphatic tissue and in the germinal centers of the secondary nodules (Fig. 6). Most of the cells in the lymphatic cords and many of those in the germinal centers were also positive for A.P. activity (Fig. 7).
Fig. 1. Two primary nodules (arrowheads) and a secondary nodule (arrow) in an ovine hemal node. x 400. Figs 2 and 3. ATPase and 5'Nase positive staining of lymphocytes making primary and the wall of secondary nodules. In addition 5'Nase stain also the dendritic reticular cells. Fig. 2, x 120; Fig. 3, x40. 299
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.
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Figs 4 and 5. Immunohistochemical evidentiation of cells bearing surface lmmunoglobulins in the sheep• Fig. 4, x 40; Fig. 5, x 250. Figs 6 and 7. Most of the mononuclear cells in the cords display a positive staining for ANAE and A.P. Fig. 6, x20; Fig. 7, x40.
300
Lymphocytes in ruminant hemal nodes
301
DISCUSSION This study shows that both primary and secondary nodules are present in hemal nodes of ruminants. The terms "primary" and "secondary" are used to designate nodules consisting entirely of small packed lymphocytes or nodules formed by large lymphoid cells and macrophages showing a light-staining germinal center, surrounded by a dark cortex [9], respectively. The presence of secondary nodules in lymphoid organs appear to be indicative for a substantial and sustained demand of antibody production [10]. The identification of lymphoid cells was mainly carried out by means of cytoenzymologic methods regarded as specific for B and T lymphocytes evidentiation by many authors [7, 11-18]. In particular Miiller-Hermelink [12] demonstrated that the distribution of EAC-binding cells in human lymph nodes was superimposable to that of 5'Nase and ATPase positive cells. Lymphatic cells in areas which did not bind EAC were also negative for 5'Nase and ATPase activity. Therefore these two enzymes can be considered specific markers for B lymphocytes. Regarding the ~-naphthyl-acetate esterase activity, many authors [7, 13, 15, 18] have reported that the ANAE staining, both in blood smears and in lymph node sections, enables the identification of the same cells as with the E-rosetting method. Therefore the ANAE staining provides a reliable estimate of the percentage of T lymphocytes. Finally the A.P. activity has been estimated to be a specific marker for T lymphocytes by Zucker-Franklin [16]. These cytochemical studies demonstrated that most of the cells in the primary nodules and in the cortex of the secondary nodules were intensely ATPase and 5'Nase positive and also expressed surface immunoglobulins. This finding suggests that these cells should be classified as B lymphocytes. On the other hand, the localization of acid hydrolases (ANAE, AP) seen in the majority of lymphoid cells of the interfollicular cords and of some germinal centers of the secondary nodules, allow to identify these cells as T lymphocytes. The monocyte-macrophage series in the last two areas displayed a diffuse cytoplasmatic reaction with the same substrate. Based on the results of this study, the morphology of the ovine and bovine hemal nodes appear to be peculiar [19-21]. Although the arrangement of the hemal node lymphatic tissue resembles that of normal lymph node, the direct contact between the lymphatic tissue and the blood is more reminiscent of the spleen. This may suggest that hemal nodes and spleen eventually perform similar functions, that is they may serve as sites where circulating antigens activate suitably programmed lymphocytes to develop into immunologically functioning cells. Acknowledgements--We thank Professor C. E. Grossi and Professor G. Castrucci for advice and criticism.
REFERENCES 1. Clarkson A. Report on hemal glands. Br. reed. J. 2, 183 (1891). 2. Robertson W. F. The prevertebral haemolymph glands. Lancet 2, 1152 (1890). 3. Badou A. C. and Rovere R. J. Los ganglios hematicos de los ruminantes. Riv. militar Vet. (Buenos Aires) 19, 51-66 (1971).
302
P. CECCARELLI et al.
4. Enriquez-Yap E. L. Cytobiological characterization of the hemolymph nodes in the Philippine carabao (Bubalus bubalis): an anatomico-histologic study on its hemopoietic and immunologic role. Philippine J. eet. Med. 14, 15 37 (1975). 5. Ceccarelli P., Gargiulo A. M., Fagioli O. and Lorvik S. Identification of lymphocytes extracted from bovine hemal nodes. Bas. appl. Histochem. 26, 259-266 (1982). 6. Wachstein M. and Meisel E. Histochemistry of hepatic phosphatases at a physiologic pH with special reference to the demonstration of bile canaliculi. Am. J. Clin. Path. 27, 13 23 (1957). 7. Giorno R. and Beverly S. A rapid method for determining T lymphocyte levels using acid alfa-naphthylacetate-esterase. Stain Technol. 56, 189 193 (1981). 8. Sternberger L. A. lmmunocytochemistry. Wiley, New York (1979). 9. Weiss L. The Blood Cells and Hematopoietic Tissues. McGraw Hill, New York (1977). 10. Ham H. W. and Cormack D. H. Histology. Lippincott, Philadelphia (1979). I I. Li C. Y., Yam L. T. and Lam K. W. Acid phosphatase isoenzyme in human leukocytes in normal and pathologic conditions. J. Histochem. Cytochem. 18, 473-480 (1970). 12. Mfiller-Hermelink H. K. Characterization of the B-cell and T-cell regions of human lymphatic tissue through enzyme histochemical of ATPase and 5'nucleotidase activities. Virchows Arch. B. cell. Path. 16, 371 378 (1974). 13. Mueller J., Brun Del Re G., Buerki H., Keller H. U., Hess M. W. and Cottier H. Nonspecific acid esterase activity: criterion for differentiation of B and T lymphocytes in mouse lymph nodes. Eur. J. lmmun. 5, 270-274 (1975). 14. Knowles D. H., Hoffman T., Ferrarini M. and Kunkel H. G. The demonstration of acid naphthyl-acetateesterase activity in human iymphocytes: usefulness as a T-cell marker. Cell. Immun. 35, 112 123 (1978). 15. Pinkus G. S., Hargreaves H. K., McLeod J. A., Nadler L. M., Rosenthal D. S. and Said J. W. Naphthyl acetate esterase activity. A cytochemical marker for T Lymphocytes. Am. J. Path. 27, 17-42 (1979). 16. Zucker-Franklin D., Greaves M. F., Grossi C. E. and Marmont A. M. Le Cellule del Sangue, Vol. 2, Chap. 9. Ermes, Milan (1981). 17. Dhingra V. K., Gupta R. K. P. and Sadana J. R. Demonstration of alpha naphthyl acetate esterase activity in bovine lymphocytes and monocytes or macrophages. Res. vet. Sci. 33, 26-30 (1982). 18. Kajikawa O., Koyama H., Yoshikawa T., Tsubaki S. and Saito H. Use of alpha-naphthyl acetate esterase staining to identify T. Lymphoeytes in cattle. Am. J. vet. Res. 44, 1549 1552 (1983). 19. Lorvik S., Gargiulo A. M., Fagioli O. and Ceccarelli P. I nodi ematici dell'ovino. Atti S I S V E T XXX|V, 140 (1980). 20. Lorvik S., Gargiulo A. M., Ceccarelli P. and Fagoli O. I nodi ematici del bovino. Arch. Vet. It. 34, I I0 124 (1983). 21. Folse D. S., Bethard G. A., Marshall R. B., Fish J. C., Sarles M. E., Remmers A. R. and Ritzmann S. E. Characterization of the bovine hemal node. J. reticuloend. Soc. 10, 461-481 (1971).
0147-9571/86 $3.00+0.00 Pergamon Journals Ltd
C Y T O C H E M I C A L I D E N T I F I C A T I O N OF L Y M P H O C Y T E S A N D O T H E R M O N O N U C L E A R CELLS IN O V I N E AND BOVINE HEMAL NODES P. CECCARELLI, A. M. GARGIULO, O. FAGIOLI a n d V. PEDINI Istituto di Anatomia Normale Veterinaria, Via S. Costanzo, 4 06100 Perugia, Italy Abstraet~Cytochemical and immunocytochemical studies were carried out with specific markers for B lymphocytes, dendritic reticulum cells (ATPase, 5'Nase, SIg) and T lymphocytes (ANAE, A.P.) in an attempt to identify the mononuclear cells present in bovine and ovine hemal nodes. The results show that primary nodes and mantle of secundary nodes are composed of B cells, whereas T cells are mainly localized in the interfollicular cords. Since such an arrangement resembles the picture in normal lymph nodes, but the direct contact between lymphatic tissue and blood is more reminiscent of the spleen, hemal nodes probably perform immunological functions similar to those of both normal lymph nodes and spleen.
Key words: Hemal node, ruminants, cytochemistry, immunocytochemistry, B lymphocyte, T lymphocyte
R~sum&-On a cherch6 ~iidentifier les cellules mononucl~aires pr~sentes dans les noeuds h+matiques (H.N.) des ovins et des bovins avec des m+thodes d'histochimie et d'himmunohistochimie. On a mis en 6vidence des lymphocytes B, des cellules r+ticulaires dendritiques et des lymphocytes T, en employant des marquers 6nzymatiques sp6cifiques: pour les lymphocytes B e t les cellules r&iculaires, l'ATPase, la 5'Nase et les immunoglobulines de surface; et pour les lymphocytes T, I'ANAE et I'A.P. Les lymphocytes B constituent les nodules primaires et la portion p+riph6rique des nodules secondaires; les lymphocytes T sont accumul~s surtout dans les cordons interfolliculaires. Cette disposition du tissue lymphatique ressemble h celle d'un classique noeud lymphatique mais, d'autre part, le contact direct entre le tissu lymphatique et le sang rappelle la structure de la rate. On peut ainsi conclure que les noeuds h6matiques jouent le m6me r61e immunologique que les noeuds lymphatiques et la rate.
Mots-clefs: Noeud h6matique, ruminants, histochimie, immunohistochimie, lymphocyte B, lymphocyte T
INTRODUCTION T h e f u n c t i o n o f h e m a l n o d e s , i.e. the l y m p h a t i c o r g a n s o f r u m i n a n t s r e l a t e d to the b l o o d b u t n o t to the l y m p h c i r c u l a t i o n , is still i n c o m p l e t e l y u n d e r s t o o d . A c c o r d i n g to e a r l y r e p o r t s , these h e m o l y m p h a t i c o r g a n s m i g h t be i n v o l v e d w i t h e i t h e r the e r y t h r o p o i e s i s [!] o r the m y e l o p o i e s i s [1,2]. H o w e v e r , m o r e r e c e n t l y it has b e e n s u g g e s t e d t h a t h e m a l n o d e s m a y p l a y a role in the i m m u n e r e s p o n s e [3-5]. I n p r e v i o u s studies it was s h o w n t h a t the r a t e o f B a n d T l y m p h o c y t e s in b o v i n e h e m a l n o d e s is s i m i l a r to t h a t f o u n d in the p e r i p h e r a l b l o o d [5]. T h e p r e s e n t p a p e r r e p o r t s a c y t o c h e m i c a l s t u d y w h e r e e n z y m e m a r k e r s specific for l y m p h o i d a n d m o n o c y t i c cells w e r e used in o r d e r to i d e n t i f y a n d to l o c a l i z e the cells in the h e m a l n o d e s o f r u m i n a n t s . 297 (IMID
94
A
298
P. CECCARELLI et al.
MATERIALS AND METHODS Hemal nodes were removed from 15 healthy cattle and 15 healthy sheep of both sexes, shortly after slaughter. Small fragments of each hemal node were frozen by immersion in iso-pentane pre-cooled with liquid nitrogen and then sectioned at 8 ~m in a cryostat. Cells making the parenchyma of the H.N. were identified by demonstrating the following enzymatic activities: ATPase, 5' nucleotidase (5' Nase), non-specific acid-c~-naphthylacetate esterase (ANAE) and acid phosphatase (A.P.) For ATPase and 5'Nase activity demonstration, cryostat sections were fixed for 15-30 min with 4% fresh formalin buffered with 0.1 M Na-cacodylate pH 7.2 and then incubated in a medium prepared according to Wachstein and Meisel [6]. The ANAE activity was revealed on fixed sections incubated in 50 ml of 0.1 M sodium acetate pH 5.2 containing 20 mg cc-naphthyl acetate and 15 mg fast garnet GBC [7]. The A.P. activity was revealed incubating the fixed sections in 10 ml 0.1 M acetate buffer pH 5.0 containing 2mg naphthol AS-BI phosphoric acid (dissolved in 0.1 ml N,N-dimethyl-formamide) and 10 mg fast garnet GBC. All reagents, except sodium acetate and acetic acid, were from Sigma Chemical Co. Controls were performed incubating the fixed sections in a medium without substrate. In the sheep hemal nodes the cells bearing surface immunoglobulins (SIg) have been also identified using an indirect immunoperoxidase technique according to Sternberger [8]. An anti-sheep IgG serum raised in rabbit (Miles-Yeda Ltd) has been used as first antibody. Sections incubated in a medium where first specific antibody was substituted with PBS served as controls.
RESULTS Morphological studies revealed both in bovine and ovine two types of nodules within the hemal nodes: "primary nodules" containing exclusively the small lymphocytes, and "secondary nodules" consisting of a light staining germinal center composed of large lymphoid cells and macrophages, surrounded by a darker mantle of small lymphocytes (Fig. 1). The small lymphocytes of the primary nodules and those making the wall of the secondary nodules were intensely ATPase (Fig. 2) and 5'Nase (Fig. 3) positive. Dendritic reticular cells and the endothelial-like cells that lined the blood sinus walls also displayed 5'Nase activity. Immunohistochemical studies showed that almost all the cells of the primary nodule and the mantle cells of secondary nodule, expressed SIg (Figs 4 and 5). When the mononuclear cells were subjected to the ANAE reaction, they showed two distinct staining patterns: i.e. of granular or diffuse type, respectively. According to this finding two types of cells seem to be involved. Both were localized primarily in the cords of lymphatic tissue and in the germinal centers of the secondary nodules (Fig. 6). Most of the cells in the lymphatic cords and many of those in the germinal centers were also positive for A.P. activity (Fig. 7).
Fig. 1. Two primary nodules (arrowheads) and a secondary nodule (arrow) in an ovine hemal node. x 400. Figs 2 and 3. ATPase and 5'Nase positive staining of lymphocytes making primary and the wall of secondary nodules. In addition 5'Nase stain also the dendritic reticular cells. Fig. 2, x 120; Fig. 3, x40. 299
!~
" 7 ~"
•, "
•r
n r
~' *
.
•4
Figs 4 and 5. Immunohistochemical evidentiation of cells bearing surface lmmunoglobulins in the sheep• Fig. 4, x 40; Fig. 5, x 250. Figs 6 and 7. Most of the mononuclear cells in the cords display a positive staining for ANAE and A.P. Fig. 6, x20; Fig. 7, x40.
300
Lymphocytes in ruminant hemal nodes
301
DISCUSSION This study shows that both primary and secondary nodules are present in hemal nodes of ruminants. The terms "primary" and "secondary" are used to designate nodules consisting entirely of small packed lymphocytes or nodules formed by large lymphoid cells and macrophages showing a light-staining germinal center, surrounded by a dark cortex [9], respectively. The presence of secondary nodules in lymphoid organs appear to be indicative for a substantial and sustained demand of antibody production [10]. The identification of lymphoid cells was mainly carried out by means of cytoenzymologic methods regarded as specific for B and T lymphocytes evidentiation by many authors [7, 11-18]. In particular Miiller-Hermelink [12] demonstrated that the distribution of EAC-binding cells in human lymph nodes was superimposable to that of 5'Nase and ATPase positive cells. Lymphatic cells in areas which did not bind EAC were also negative for 5'Nase and ATPase activity. Therefore these two enzymes can be considered specific markers for B lymphocytes. Regarding the ~-naphthyl-acetate esterase activity, many authors [7, 13, 15, 18] have reported that the ANAE staining, both in blood smears and in lymph node sections, enables the identification of the same cells as with the E-rosetting method. Therefore the ANAE staining provides a reliable estimate of the percentage of T lymphocytes. Finally the A.P. activity has been estimated to be a specific marker for T lymphocytes by Zucker-Franklin [16]. These cytochemical studies demonstrated that most of the cells in the primary nodules and in the cortex of the secondary nodules were intensely ATPase and 5'Nase positive and also expressed surface immunoglobulins. This finding suggests that these cells should be classified as B lymphocytes. On the other hand, the localization of acid hydrolases (ANAE, AP) seen in the majority of lymphoid cells of the interfollicular cords and of some germinal centers of the secondary nodules, allow to identify these cells as T lymphocytes. The monocyte-macrophage series in the last two areas displayed a diffuse cytoplasmatic reaction with the same substrate. Based on the results of this study, the morphology of the ovine and bovine hemal nodes appear to be peculiar [19-21]. Although the arrangement of the hemal node lymphatic tissue resembles that of normal lymph node, the direct contact between the lymphatic tissue and the blood is more reminiscent of the spleen. This may suggest that hemal nodes and spleen eventually perform similar functions, that is they may serve as sites where circulating antigens activate suitably programmed lymphocytes to develop into immunologically functioning cells. Acknowledgements--We thank Professor C. E. Grossi and Professor G. Castrucci for advice and criticism.
REFERENCES 1. Clarkson A. Report on hemal glands. Br. reed. J. 2, 183 (1891). 2. Robertson W. F. The prevertebral haemolymph glands. Lancet 2, 1152 (1890). 3. Badou A. C. and Rovere R. J. Los ganglios hematicos de los ruminantes. Riv. militar Vet. (Buenos Aires) 19, 51-66 (1971).
302
P. CECCARELLI et al.
4. Enriquez-Yap E. L. Cytobiological characterization of the hemolymph nodes in the Philippine carabao (Bubalus bubalis): an anatomico-histologic study on its hemopoietic and immunologic role. Philippine J. eet. Med. 14, 15 37 (1975). 5. Ceccarelli P., Gargiulo A. M., Fagioli O. and Lorvik S. Identification of lymphocytes extracted from bovine hemal nodes. Bas. appl. Histochem. 26, 259-266 (1982). 6. Wachstein M. and Meisel E. Histochemistry of hepatic phosphatases at a physiologic pH with special reference to the demonstration of bile canaliculi. Am. J. Clin. Path. 27, 13 23 (1957). 7. Giorno R. and Beverly S. A rapid method for determining T lymphocyte levels using acid alfa-naphthylacetate-esterase. Stain Technol. 56, 189 193 (1981). 8. Sternberger L. A. lmmunocytochemistry. Wiley, New York (1979). 9. Weiss L. The Blood Cells and Hematopoietic Tissues. McGraw Hill, New York (1977). 10. Ham H. W. and Cormack D. H. Histology. Lippincott, Philadelphia (1979). I I. Li C. Y., Yam L. T. and Lam K. W. Acid phosphatase isoenzyme in human leukocytes in normal and pathologic conditions. J. Histochem. Cytochem. 18, 473-480 (1970). 12. Mfiller-Hermelink H. K. Characterization of the B-cell and T-cell regions of human lymphatic tissue through enzyme histochemical of ATPase and 5'nucleotidase activities. Virchows Arch. B. cell. Path. 16, 371 378 (1974). 13. Mueller J., Brun Del Re G., Buerki H., Keller H. U., Hess M. W. and Cottier H. Nonspecific acid esterase activity: criterion for differentiation of B and T lymphocytes in mouse lymph nodes. Eur. J. lmmun. 5, 270-274 (1975). 14. Knowles D. H., Hoffman T., Ferrarini M. and Kunkel H. G. The demonstration of acid naphthyl-acetateesterase activity in human iymphocytes: usefulness as a T-cell marker. Cell. Immun. 35, 112 123 (1978). 15. Pinkus G. S., Hargreaves H. K., McLeod J. A., Nadler L. M., Rosenthal D. S. and Said J. W. Naphthyl acetate esterase activity. A cytochemical marker for T Lymphocytes. Am. J. Path. 27, 17-42 (1979). 16. Zucker-Franklin D., Greaves M. F., Grossi C. E. and Marmont A. M. Le Cellule del Sangue, Vol. 2, Chap. 9. Ermes, Milan (1981). 17. Dhingra V. K., Gupta R. K. P. and Sadana J. R. Demonstration of alpha naphthyl acetate esterase activity in bovine lymphocytes and monocytes or macrophages. Res. vet. Sci. 33, 26-30 (1982). 18. Kajikawa O., Koyama H., Yoshikawa T., Tsubaki S. and Saito H. Use of alpha-naphthyl acetate esterase staining to identify T. Lymphoeytes in cattle. Am. J. vet. Res. 44, 1549 1552 (1983). 19. Lorvik S., Gargiulo A. M., Fagioli O. and Ceccarelli P. I nodi ematici dell'ovino. Atti S I S V E T XXX|V, 140 (1980). 20. Lorvik S., Gargiulo A. M., Ceccarelli P. and Fagoli O. I nodi ematici del bovino. Arch. Vet. It. 34, I I0 124 (1983). 21. Folse D. S., Bethard G. A., Marshall R. B., Fish J. C., Sarles M. E., Remmers A. R. and Ritzmann S. E. Characterization of the bovine hemal node. J. reticuloend. Soc. 10, 461-481 (1971).