Vet. Pathol. 20: 322-329 (1983) Cytochemical Reactions in Bovine and Ovine Lymphosarcoma P. C. RAICH, I. TAKASHIMA, and C. OLSON Division of Hematology, Department of Medicine and Department of Veterinary Science, University of Wisconsin, Madison, Wisc. and Section of Hematology/Oncology, Department of Medicine, West Virginia University, Morgantown, W. Va. Abstract. Cytochemical reactions with a panel of special stains were explored in peripheral blood and lymph node cells of animals with the adult, calf, and thymic types of bovine lymphosarcoma as well as lymphosarcoma in sheep induced with the bovine leukosis virus. These results were compared with normal lymphoid cells. Cytochemical methods included Sudan black B, periodic acid-schiff, acid phosphatase and three esterases. Cells from ovine lymphosarcomas and a thymic type of bovine lymphosarcoma were distinctively positive to the alpha naphthyl acetate esterase reaction. These cytochemical reactions which were obtained with standard cytochemical methods should aid in the characterization and classification of lymphoproliferative disorders in animals, especially when combined with immunologic surface markers. Cytochemical reactions have been studied extensively in leukemias and lymphoproliferative disorders in man 12, 3, 191. No similar studies have been reported in bovine and ovine lymphosarcoma. In previous studies findings have been reported of B and T cell typing in three types of bovine lymphosarcoma [17] and surfaceimmunoglobulin-bearing cells in sheep infected with bovine leukosis virus [ 161. This report expands these observations to include cytochemical reactions in three of the four types of bovine lymphosarcoma and in ovine lymphosarcoma induced with bovine leukosis virus. Materials and Methods The peripheral blood lymphocytes and/or lymph nodes from nine clinically and macroscopically normal cattle and two sheep were obtained from an abattoir. These animals were negative for both bovine leukosis virus and bovine leukosis virus antibody, as determined by lymphocyte culture and double gel immunodiffusion, respectively [ 171. Nine cattle with the adult type of lymphosarcoma were obtained from the same abattoir; they were three years of age or older and had scattered lymph nodes enlarged with lymphosarcoma. These cattle demonstrated antibodies against bovine leukosis virus. Two cattle with the calf type of lymphosarcoma were obtained from another abattoir. Tumorous thymus and blood from one thymic lymphosarcoma were provided by Dr. W. Jarrett, University of Glasgow, Scotland. 322
Cytochemistry in Lymphosarcoma 323 Peripheral blood lymphocytes for study also were obtained from five experimentally bovine leukosis virus-infected sheep with lymphosarcoma. Peripheral blood smears and imprints from normal and lymphosarcoma lymph nodes and thymus were stained with modified Wright s stain and with the following special cytochemical reactions according to previously published methods: Sudan black (SBB) [7], periodic acid- Schiff (PAS) [ I], combined naphthol AS-D chloracetate (NASDC) and alpha naphthyl acetate (ANA) esterase [ 181, naphthol AS-D acetate (NASDA) esterase with and without sodium fluoride [14], and acid phosphatase (ACP) with and without tartrate inhibition [8]. The activity of the staining reactions in the normal lymphocytes and lymphosarcoma cells was scored according to criteria defined previously 121 over a range of 0 to 3+, with 0 representing no staining, and 3+ the strongest reaction. In addition, the percentage of positive cells for each staining reaction was tabulated. B and T cell studies: Methods for lymphocyte preparation, surface immunoglobulin labeling, erythrocyte antibody complement and E-rosettes were done according to methods previously outlined [ 16, 171. Results Both peripheral blood lymphocytes and cells from lymph node imprints were uniformly negative for SBB, PAS, and NASDC esterase reactions. Fifty-six percent of peripheral blood lymphocytes stained positive with ANA esterase in a predominantly focal pattern, while only 15% of lymph node lymphocytes stained positive with ANA esterase. The majority of peripheral blood and lymph node lymphocytes stained positive with NASDA esterase which was affected little by fluoride. Fewer cells reacted positively with acid phosphatase and all of this activity was inhibited by tartrate incubation. In one animal, cells from lymph nodes from four different anatomical sites demonstrated identical cytochemical reactions. Twenty percent of blood lymphocytes and lymph node cells were surface immunoglobulin positive (table I). Cytochemical reactions from fetal peripheral blood and lymph node preparations were similar to the corresponding adult tissue. Fetal thymus cells reacted similarly to lymph nodes. Twenty percent of fetal lymph node cells and no thymus cells were surface immunoglobulin positive. Reactions of cells from both the adult and the calf type of lymphosarcoma closely resembled those of normal bovine lymphoid tissue. SBB, PAS, and NASDC esterase reactions were negative. ANA esterase reaction was moderately positive in less than 45% of cells. NASDA esterase reaction was weakly positive with partial inhibition by fluoride. A larger percent of adult type lymphosarcoma cells reacted positively with ACP (83%). Most of this activity was lost upon incubation with tartrate. Both peripheral blood lymphocytes and thymic cells from an animal with the thymic type of lymphosarcoma reacted positively with ANA esterase, with a strong and dense reaction in almost all cells (fig. 1). Again SBB, PAS, and NASDC esterase reactions were negative, and ACP reaction positive in 30%. Lymphocytes on lymph node imprints from two normal sheep had negative reactions when stained with SBB, PAS, and NASDC esterase. ANA esterase reaction
~ Table I. Cytochemical findings in bovine and ovine normal and lymphosarcoma cells Cytochemical reactions* (% cells positive) 0 N P Tissue Bovine, normal Adult Fetal THY Bovine lymphosarcoma Adult type Calf type Thymic type THY Ovine, normal Ovine lymphosarcoma Number B Cells Cases (%) SBB PAS NASDCE ANAE 6 50 - f (3) 7 70 - - 1 1.5 - - 2 0 - - NASDAE ACP 1 10 - - - +++ (86) ++ (75) f ( 7) 1 0 - - - +++ (98) plain NAF plain Tartrate + (73) (63) + (30) - + (53) + (46) + (15) - (61) (59) E (44) - + 6 (55) (62) (37) f (10) > ++ (97) + (77) + ( 5) - P 2 ND - - - + (7) + (36) + (15) 5 94 - - - +++ (93) ++ (83) f (30) ++ (89) f (25) * See text for abbreviations of cytochemical reactions. = peripheral blood lymphocytes; = lymph node cells; THY = thymus cells; +++ = strong solid or block-like positive; ++ = strong positive; + = moderately positive; +_ = weakly positive; - = negative.
Cytochemistry in Lymphosarcoma 325 Fig. 1: Cytocentrifuge preparation of thymus cells from bovine thymic lymphosarcoma with dense cytoplasmic positive reaction. Alpha naphthyl acetate esterase. Bar = 20 pm. Fig. 2 Peripheral blood smear from ovine lymphosarcoma with focal positive reaction. Alpha naphthyl acetate esterase. Bar = 20 pm. was positive in only a few cells, and a positive ACP reaction was noted in 30% of cells with partial loss of activity with tartrate incubation. The peripheral blood lymphocytes from five sheep with lymphosarcoma induced by bovine leukosis virus had distinctive cytochemical reactions, which differed from normal lymphocytes and from the adult and fetal bovine lymphosarcomas, but had some similarities with the thymic type. Again SBB, PAS, and NASDC esterase reactions were negative in the abnormal lymphocytes. As in the bovine thymic lymphosarcoma, ANA esterase reaction was strongly positive in virtually all cells; however, there was a multi-focal, large granular or block-like positive reaction (fig. 2), rather than the diffuse cytoplasmic positive reaction seen in the bovine thymic lymphosarcoma. Acid phosphatase reaction was moderately positive in most cells, and this activity was inhibited partially by tartrate incubation. It is of interest that 87 to 97.5% of the peripheral blood mononuclear cells were surface immunoglobulin
326 Raich, Takashima, and Olson positive in the sheep while only 10% of peripheral blood lymphocytes were surface immunoglobulin positive in the bovine thymic lymphosarcoma. Discussion The use of cytochemical staining methods has aided considerably in the classification of acute leukemias in man [6]. Along with B and T lymphocyte typing, cytochemical reactions also have allowed for better characterization of a variety of lymphoproliferative disorders in man [ 101. In bovine lymphosarcoma, previous studies [I71 have shown that the adult type may have B cell characteristics (increased number of cells with surface immunoglobulin and erythrocyte antibody complement receptors in both peripheral blood and lymph nodes), while cells of the calf and thymic types could not be characterized as either B or T and may represent a malignant transformation of lymphocyte stem cells, or may represent pre-b cells, similar to the human null -type acute lymphocytic leukemia. Sheep experimentally infected with bovine leukosis virus develop lymphosarcoma with a high frequency over an average time of 29 months [12]. The peripheral blood lymphosarcoma cells in these sheep are largely B cells [16]. In order to assess the usefulness of cytochemical staining reactions in addition to the immunological markers toward the characterization of lymphoproliferative disorders in animals, the cytochemical reactions of normal cells need to be characterized. Lymphocytes from normal fetal and adult bovine peripheral blood, lymph nodes from various sites, fetal thymus and ovine lymph nodes had consistent cytochemical reactions. ANA esterase and NASDA esterase with and without fluoride and acid phosphatase reactions were mildly to moderately positive, with acid phosphatase losing its activity with tartrate incubation. These reactions are comparable to those observed in normal human blood and tonsillar lymphocytes [13]. ANA esterase reactivity was found to be an excellent marker for T cells with considerably better specificity than acid phosphatase [ 131. Our results on normal bovine lymphocytes also indicate a larger percentage of ANA esterase positive cells in the peripheral blood as compared with lymph nodes, reflecting the larger T cell population in the peripheral blood. The cytochemical reactions observed in the bovine adult lymphosarcomas were similar to those seen in the corresponding normal tissues. If indeed the malignant cells are derived from a relatively well-differentiated B cell such similar reactions would be expected. The staining reactions with SBB, PAS, and NASDC esterase were negative, confirming the non-granulocytic and non-monocytic origin of these cells. Lymphosarcoma in man (lymphocytic lymphoma, poorly differentiated, and histiocytic lymphoma) and chronic lymphocytic leukemia, which occur usually in the adult, also are predominantly B cell lymphoproliferative disorders [5, 101. The cytochemical reactions in these disorders are similar to those observed in the adult type bovine lymphosarcoma [9, 111. The two calf type bovine lymphosarcomas examined had a very low percentage of
Cytochernistry in Lymphosarcoma 321 B cells (as determined by surface immunoglobulin and erythrocyte antibody complement-rosettes)- 1.5 and 0%-and T-cells (E-rosettes)-4 and 2%-in the peripheral blood and lymph nodes, respectively [ 171. The cytochemical reactions observed were relatively nonspecific and resembled both the normal tissues and the adult bovine lymphosarcomas. The human counterpart of this subtype is approached most closely by the common or null cell variety of acute lymphocytic leukemia, which occurs most often during childhood and in young adults [6]. Here the leukemic cells are also neither B or T cells by standard marker studies. Aside from PAS reaction, which may show a characteristic block-like cytoplasmic positivity in the leukemic cells of approximately 50% of persons with acute lymphocytic leukemia, the other reactions are similar to those observed in the calf type bovine lymphosarcoma. Only one thymic bovine lymphosarcoma was available for study with cytochemical stains. No surface immunoglobulin positive cells were found in the thymus; previous studies have shown 0 to 10% E-rosette positive cells in the peripheral blood and thymus of two animals [17]. The tumor cells were small to medium lymphocytes with relatively sparse cytoplasm, at times showing a hand-mirror appearance which has been observed in some human acute lymphocytic leukemias. The cytochemical reactions were similar to those observed in the other types of bovine lymphosarcoma with the exception of a markedly positive ANA esterase reaction in 98% of the thymic cells and a large percentage of the mononuclear cells in the peripheral blood. Such a strong and dense cytoplasmic positivity has not been observed previously in normal or malignant lymphocytes and is more characteristic of monocytes [ 181. However, the morphologic characteristics and the lack of positivity with SBB and NASDC esterase point to a cell of lymphocytic origin. Acid phosphatase was positive; this is a feature of human T cell acute lymphocytic leukemia also [4]. It is of considerable interest that the lymphosarcoma induced in sheep by infection with bovine leukosis virus also is associated with an unusual and strong ANA esterase reaction in the malignant lymphocytes, but rather than showing a solid positive reaction throughout the entire cytoplasm, the cells feature focal, spot-like areas of unequal size scattered throughout the entire cytoplasm, usually three to ten per cell (fig. 2). The morphology of the tumor cells also differs considerably from the thymic bovine lymphosarcoma. In the ovine type the cells are large with plentiful cytoplasm. A high percentage (average 94%) were found to possess surface immunoglobulin. Two human disorders of probable lymphocytic origin, hairy cell leukemia (leukemic reticuloendotheliosis) and a variant form of acute lymphocytic leukemia [15], also have a moderate to strong reaction with ANA esterase. Hairy cell leukemia cells, in addition, usually have a strongly positive acid phosphatase reaction which, unlike all other normal and malignant cells, is resistant to incubation with tartrate [14]. A strong acid phosphatase reaction was seen in 89% of the tumor cells of sheep with lymphosarcoma but most of this activity was not resistant to tartrate exposure. The variant of acute lymphocytic leukemia [15] had strong reactions with ANA esterase and NASDA esterase, and the latter was not affected by sodium fluoride
328 Raich, Takashima, and Olson incubation. No acid phosphatase staining and B and T cell typing were done. Certain features of this human leukemia variant are reminiscent of the T cell type of acute lymphocytic leukemia, although T cells (both normal and leukemic) usually feature a single spot of ANA esterase positivity. The application of cytochemical reactions to bovine and ovine bovine leukosis virus-related lymphosarcoma has demonstrated unique and specific findings among certain subtypes of bovine lymphosarcoma and with the bovine leukosis virusinduced disease in sheep. These reactions, in addition to T and B cell typing, should be useful in the classification of lymphoproliferative disorders in these animalssimilar to that established in man [lo]. The ANA esterase reaction was striking in the thymic bovine lymphosarcoma. Cytochemical investigations of additional animals with this rare bovine lymphosarcoma are needed to substantiate this finding. The ANA esterase reaction also was consistently positive with a unique pattern in all ovine lymphosarcomas. Certain similarities also can be observed in comparison with lymphoproliferative disorders and lymphoreticular neoplasms of man, where these markers have been useful in establishing a classification system with strong implications to prognosis and treatment. Acknowledgements We appreciate the excellent technical assistance of Mrs. Connie Arndt, Mrs. Mary Gallegly, Mr. Randall Kaja, and Mr. Robert Mertz, and the secretarial assistance of Ms. Annorah Cale. I. Takishima s present address is: Faculty of Veterinary Medicine, Hokkaido Univ., Sapporo, 060 Japan. References 1 ARMED FORCES INSTITUTE of PATHOLOGY: Manual of Histologic and Special Staining Technics, 2nd ed.. McGraw Hill Book Co., Inc., New York, 1960 2 BENNETT, J.M.; REED, C.E.: Acute leukemia cytochemical profile: Diagnostic and clinical implications. Blood cells 1: 101-108, 1975 3 CATOVSKY, D.; GALLETTO, J.; OKOS, A,; MILIANI, E.; GALTON, D.A.G.: Cytochemical profile of B and T leukemic lymphocytes with special reference to acute lymphoblastic leukemia. J Clin Pathol27767-771, 1974 4 CATOVSKY, D.; GREAVES, M.F.; PAIN, C.; CHERCHI, M.; JANOSSY, G.; KAY, H.E.M.: Acidphosphatase reaction in acute lymphoblastic leukemia. Lancet 1:749-75 1, 1978 5 GAJL-PECZALSKA, K.J.; BLOOMFIELD, C.D.; COCCIA, P.R.; SOSIN, H.; BRUNNING, R.D.; KERSEY, J.H.: B and T cell lymphomas: Analysis of blood and lymph nodes in 87 patients. Am J Med 59674-685, 1975 6 GRAICK, J.R.; GALTON, D.A.G.; CATOVSKY, D.; SULTAN, C.; BENNETT, J.M.: Classification of acute leukemia. Ann Intern Med 87:740-753, 1977 7 HAYHOE, F.G.J.; FLEMANS, R.J.: Atlas of Haemotological Cytology, p. 316. Wiley and Sons, Inc., New York, 1970 8 KATAYAMA, I.; LI, C.Y.; YAM, L.T.: Histochemical study of acid phosphatase isoenzymes in leukemic reticuloendotheliosis. Cancer 29157-164, 1972 9 LI, C.Y.; LAM, K.W.; YAM, L.T.: Esterase in human leukocytes. J Histochem Cytochem 21: 1-12, 1973
Cytochemistry in Lymphosarcoma 329 10 LUKES, R.J.: COLLINS, R.D.: New approaches to the classification of the lymphomata. Br J Cancer (Suppl) 31: 1-28, 1975 11 MORRIS, M.W.; DAVEY, F.R.: Immunologic and cytochemical properties of histiocytic and mixed histiocytic-lymphocytic lymphomas. Am J Clin Pathol 63:403-414, 1975 12 OLSON, C.; MILLER, L.D.; MILLER, J.M.; HON, H.E.: Transmission of lymphosarcoma from cattle to sheep. JNCI 491463-1467, 1972 13 PANGALIS, G.A.; WALDMAN, S.R.; RAPPAPORT, H.: Cytochemical findings in human nonneoplastic blood and tonsillar B and T lymphocytes. Am J Clin Pathol 69314-318, 1978 14 ROSENSZAJN, L.A.; GURMAN, A.; RADNAY, J.; DAVID, E.B.; SHOHAM, D.: A study of the nature of hairy cells, with emphasis on enzymatic markers. Am J Clin Pathol 66432-441, 1976 15 SHAW, M.T.; ISHMAEL, D.R.: Acute lymphocytic leukemia with atypical cytochemical features. Am J Clin Pathol63415-420, 1975 16 TAKASHIMA, I.; OLSON, C.: Bovine leukosis virus in sheep, lymphocyte modification and surface-immunoglobulin-bearing cell numbers. Vet Microbiol 5 1-12, 1980 17 TAKASHIMA, I.; OLSON, C.; Driscoll, D.M.; BAUMGARTENER, L.E.: B-lymphocytes and T- lymphocytes in three types of bovine lymphosarcoma. JNCI 591205-1209, 1977 18 YAM, L.T.; LI, C.Y.; CROSBY, W.H.: Cytochemical identification of monocytes and granulocytes. Am J Clin PatholS5283-290, 1971 19 YAM, L.T.; TAVASSOLI, M.; JACOBS, P.: Differential characterization of the reticulum cell in lymphoreticular neoplasms. Am J Clin Pathol 64: 171-179, 1975 Request reprints from Peter C. Raich, M.D., Section of Hernatology/Oncology, Room 4096, Basic Science Building, West Virginia University Medical Center, Morgantown, WV 26506 (USA).