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Cytochemical Findings in Human Nonneoplastic Blood and Tonsillar B and T Lymphocytes GERASSIMOS A. PANGALIS, M.D., STEPHEN R. WALDMAN, PH.D., AND HENRY RAPPAPORT, M.D. Pangalis, Gerassimos A., Waldman, Stephen R., and Rappaport, Henry: Cytochemical findings in human nonneoplastic blood and tonsillar B and T lymphocytes. Am J Clin Pathol 69: 314-318, 1978. The cytochemical profiles of B and T lymphocytes from the bloods of eight normal donors and the tonsils of three normal individuals were studied. An intense and localized a-naphthyl acetate esterase (a-NAE) activity was found in the majority of blood and tonsillar T lymphocytes, in contrast to the very low a-NAE activity observed in the blood and tonsillar B lymphocytes. A very low percentage of tonsillar B lymphocytes had /3-glucuronidase (/3G) activity, while relatively normal /3G activity was observed in the tonsillar T lymphocytes and the blood B and T lymphocytes. Acid phosphatase (AcP) activities were found to be similar in both B and T lymphocytes from blood and tonsils. These findings suggest that the a-NAE reaction may be useful as a cytochemical marker for distinguishing B from T lymphocytic proliferations. They also revealed that there is no appreciable difference in AcP and /3G activity between B and T lymphocytes obtained from the blood of normal donors. (Key words: Blood; Tonsillar B and T lymphocytes; Cytochemistry; aNaphthyl acetate esterase; /^-Glucuronidase; Acid phosphatase). ACID PHOSPHATASE POS1TIVITY, particularly when localized in the Golgi zone, has been considered characteristic of T-lymphoblastic proliferations, 6 ' 719 - 20 and T-cell chronic prolymphocytic leukemia. 5 Increased /3-glucuronidase activity has been reported to be consistently present in T-cell chronic lymphocytic leukemia (CLL) and in Sezary's cells, 4,10 and decreased or absent in the cells of lymph-node imprints from diffuse non-Hodgkin's lymphomas. 16 A negative periodic acid-Schiff (PAS) reaction has been reported to occur in T-cell chronic prolymphocytic leukemia, in contrast to the B-cell CLL, in which the PAS reaction is usually positive. 5 Whether these cytochemical differences reflect characteristics of neoplastic lymphoid cells independent of their B and T derviation or are properties inherent to the B or T nature of these cells is not known. It Department of Anatomic Pathology, City of Hope National Medical Center, Duarte, California was for this reason that we decided to investigate whether differences in the cytochemical profiles of normal B and T lymphocytes do in fact exist. For this purpose we prepared smears rich in either B or T lymphocytes from blood and tonsils obtained from normal donors, on which a number of cytochemical tests were performed. Materials and Methods B and T Lymphocyte Separation Tonsils were obtained from three individuals who had tonsillectomy because of recurrent tonsillitis. Immediately after removal, the tonsils were finely minced in RPMI-1640 medium. The minced tissue and supernatant were then filtered through a stainless steel mesh, and the cells were resuspended in RPMI-1640 medium. Blood leukocytes were obtained from eight normal donors, using the Aminco "celltrifuge" continuousflow blood cell separator,* as described by Waldman and associates. 23 Tonsillar and blood mononuclear cells were then separated using the Ficoll-Hypaque technic. The interphase layer containing lymphocytes and monocytes was collected, washed, and resuspended in RPMI-1640 medium. Monocytes were eliminated by placing the cell suspensions in glass petri dishes and allowed to stand for 2 h in a 5% C 0 2 atmosphere at 37 C. Subsequently, lymphocytes were removed, recovery was determined, and cell number was adjusted to 1 x 107 /ml for the rosetting procedure. For the separation of the B and T lymphocyte population, the spontaneous sheep erythrocyte (SRBC) rosette formation technic (E rosettes) described by Abdou and colleagues 1 was employed. E rosettes were formed by incubating 2-ml volumes of 1 x 107 cells, 2 ml 1% SRBC, and 1 ml human AB serum (preadsorbed with SRBC) in a 15-ml plastic tube for 15 min at 37 C. The tubes were subsequently centrifuged at 250 x g* for 10 min and allowed to stand at 4 C for 60 min. The cell pellets were then resuspended carefully and a 4-ml Received July 28, 1977; received revised manuscript October 24, 1977; accepted for publication October 24, 1977. Supported by Grant Number R 10 CA 18044-01 awarded by the National Cancer Institute, DHEW and by Hematopathology Tutorials, Inc. Dr. Pangalis was in receipt of a fellowship from the State Scholarships Foundation of Greece. Address reprint requests to Dr. Rappaport, Department of Anatomic Pathology, City of Hope National Medical Center, Duarte, California 91010. * American Instrument Co., Silver Springs, Maryland. 0002-9173-78-0300—0314 $00.75 © American Society of Clinical Pathologists 314 vol. 69 . No. 3 315 CYTOCHEMISTRY OF B & T LYMPHOCYTES Table I. Surface Marker Studies of B-rich and T-rich Lymphocyte Populations B-lymphoc yte Rich A. Normal blood Donor 1 Donor 2 Donor 3 Donor 4 Donor 5 Donor 6 Donor 7 Donor 8 MEAN ± SE B. Tonsilar Donor 1 Donor 2 Donor 3 E-Rosettes (% of Positive Cells) Immunofluorescence (% of Positive Cells) E-Rosettes (% of Positive Cells) Immunofluorescence (% of Positive Cells) 10 7 4.5 3 10 7 13 10 8.1 ± 1.2 80 85 81 85 90 87 86 85 84.9 ± 1.1 94.5 94 90 97 94 96 89 91 93.2 ± 1.0 8 9 1 0 3 1 4 2 6.0 ± 2.5 9 10 3 85 86 90 94 97 95 7 10 4 volume of Ficoll-Hypaque precooled to 4 C, was underlayed. Following centrifugation at 700 x g* for 35 min at 10 C, the cells of the interface layer and the pellet were collected separately. The cells of the pellet were incubated in 0.84% NH..CI for 5 min at 37 C to eliminate the SRBC. Both interphase layer and pellet lymphocytes were subsequently washed twice and resuspendedin RPMI-1640 medium. Viability exceeded 90% in all cases as determined by trypan blue stain. Determinations of the composition of lymphocyte populations in the interphase layer and the pellet were performed by enumerating E rosettes 18 and detecting surface immunoglobulins by the immunofluorescence technic described by Pernis and co-workers, 17 using a polyvalent rabbit antihuman immunoglobulin labeled with fluorescein isothiocyanate.t Cytochemical T-lymphocyte Rich Study Smears of the B and T lymphocyte-rich populations from the tonsils and the blood were made on glass slides and the following cytochemical determinations were performed: Acid phosphatase (AcP)2 and acid phosphatase with tartaric acid, 12 /3-glucuronidase (J3G),11 esterase with a-naphthyl acetate as substrate (a-NAE), 26 esterase with naphthol AS-D acetate as substrate (NASDA), NASDA with sodium fluoride (NASDA + NaF),°and periodic acid-Schiff (PAS) reaction. The end products of AcP, /3G, and NASDA reactions were semiquantitatively estimated according to the extent of reaction positivity in each of at least 100 consecutively graded lymphocytes.3-21-27 a-NAE positivity was graded zero (0) when the reaction was entirely negative; 1+ when a focal positive material was present; 2 + t Cappel Laboratories, Downingtown, Pennsylvania. when 2 - 8 granules were present; 3+ when more than eight positive granules were found. One hundred lymphocytes were consecutively graded according to a-NAE reaction positivity. Results were expressed as percentages of positive cells and as scores of positivity per 100 lymphocytes. Statistical analysis of our findings was performed using the t test. Results Surface Markers The results of surface marker studies of B lymphocyte-rich and T lymphocyte-rich populations of the blood and the tonsillar specimens are enumerated in Table \,A, B. Purities of B lymphocytes of the B cellrich preparations ranged from 80 to 90%, while purities of T lymphocytes of the T cell-rich preparations ranged from 89 to 97%. The B cell-rich lymphocyte populations contained small percentages (3-13%) of T lymphocytes, presumably a few lymphocytes that were classified as B because they had bound IgG on their surfaces through a complement receptor, 25 and a small number of "null" lymphocytes that were separated with the B cell population due to their inability to form E rosettes with SRBC. Cytochemical Findings in Blood B and T Lymphocyte-rich Populations The end product of the cytochemical reactions was in the form of granules, varying in number from cell to cell. The percentages of cells containing AcP and j8G, the overall AcP and /3G activities expressed as scores, and the patterns of positivity (scattered gran- 316 PANGALIS, WALDMAN, AND RAPPAPORT A.J.C.P. . March 1978 Table 2. Results of Cytochemical Studies of Normal Blood B-rich and T-rich Lymphocyte Populations B Lymphocyte-•rich Population %* of Positive Cells Acid phosphatase /3-Glucuronidase a-Naphthyl acetate esterase Naphthol AS-D acetate esterase 45 49 30 98 ± ± ± ± 17 23 15 2 T Lymphocyte- rich Population Score* per 100 Cells 70 137 31 168 ± ± ± ± P Values of B vs. T lymphocyte-rich Populations %* of Positive Cells 33 76 15 10 61 72 71 99 Score* per 100 Cells ± 17 ± 18 ± 6 ± 2 100 138 74 171 ± ± ± ± 47 59 10 5 % Score .068 .049 .0001 .3 .14 .95 .0001 .4 * Mean * SE. a-NAE and NASDA activities with and without NaF inhibition in the tonsillar B and T lymphocyte-rich populations were found to be similar to those observed in the blood lymphocytes. In both blood and tonsillar lymphocytes, the PAS reaction was very difficult to interpret because the end product of the reaction was in a weak diffuse form rather than granular. This is probably due to the rapid change in the cytoplasmic glycogen content that occurs during incubation. 5,24 ules) were similar in the two cell populations (Table 2). Acid phosphatase activity was entirely inhibited in both B and T lymphocyte-rich populations after addition of tartaric acid. In all specimens rich in B lymphocytes, a-NAE activity was found to be present in only a small percentage of cells (Fig. 1). In contrast, in all specimens rich in T lymphocytes, a-NAE positivity was found to be present in the majority of the cells; the end product of the reaction was very intense and was localized in a small paranuclear area (Fig. 2). Statistical analysis of our findings in the blood lymphocytes showed that the difference in the a-NAE reactions between B and T lymphocyte-rich populations was highly significant (P = .0001) (Table 2). NASDA positivities were similar in the two lymphocyte populations, and the reaction was not influenced by sodium fluoride. Discussion One of the most striking findings of our study was the difference in a-NAE activities between the B and T lymphocytes. Alpha-naphthyl acetate esterase activity was found to be consistently present in an intensely localized form in blood and tonsillar T lymphocytes, in contrast to a low activities in blood and tonsillar B lymphocytes. Similar differences have been observed by Mueller and associates 13 in experimental animals. In an excellent study in which they combined immunologic, cytochemical and histochemical methods, these investigators found very low a-NAE activity in mouse B-lymphoid cells. They postulated that a-NAE might be used as a simple marker for differentiating B and T lymphocytes. The consistency, frequency and pattern of positivity with which the a-NAE reaction was observed in the T human lymphocytes indicates that further study of this reaction should be carried out in the lymphoid malignancies in conjunction with im- Cytochemical Findings in Tonsillar B and T. Lymphocyte-rich Populations AcP activities of the tonsillar B and T lymphocyterich populations were similar to those of the blood B and T lymphocyte-rich populations (Tables 2 and 3); the enzymatic activity was entirely inhibited by tartaric acid. /3-Glucuronidase activity of the tonsillar B lymphocyte-rich population was found to be present in a very small percentage of the cells, in contrast to the high percentages of positive cells in the tonsillar T lymphocyte-rich and the blood B and T lymphocyterich populations (Tables 2 and 3, and Figs. 3 and 4). Table 3. Results of Cytochemical Studies of Tonsillar B-rich and T-rich Lymphocyte Populations* T Lymphocyte-rich Population B Lymphocyte-rich Population /3G AcP a-NAE /3G AcP NASDA Donor % Score % Score % Score % Score % 1 2 3 53 65 60 86 100 96 7 8 5 10 16 9 21 24 30 21 24 30 96 98 94 154 175 168 58 69 60 • Score 98 86 101 a-NAE NASDA % Score % Score % Score 53 67 42 108 116 78 59 64 60 59 64 60 98 95 97 172 164 180 ' AcP = acid phosphatase; /3G =0-glucuronidase; a-NAE = a-naphthyl acetate esterase; NASDA = naphthol AS-D acetate esterase. 317 CYTOCHEMISTRY OF B & T LYMPHOCYTES Vol. 69 • No. 3 • • w FIG. I (upper, left). B lymphocytes from blood of a normal donor, showing a negative a-naphthyl acetate esterase reaction in most cells. An a-naphthyl acetate esterase-positive cell is present (arrow), x 1,200. FIG. 2. (upper, right). T lymphocytes from blood of the same normal donor, showing a-naphthyl acetate esterase positivity in an intense localized form in all cells, x 1,200. FIG. 3 (lower, left). B lymphocytes from tonsil of a normal donor, showing a negative /3-glucuronidase reaction in most cells. An occasional /3-glucuronidase-positive lymphocyte was observed, x 1,200. FIG. 4 (lower, right). T lymphocyte from tonsil of the same normal donor, showing a positive /3-glucuronidase reaction in most cells. This reaction is intense, with many well-defined granules. Cells with various degrees of positivity are present, x 1,200. munologic markers in an attempt to investigate the possible value of this reaction in distinguishing B from T lymphocytic proliferations. Catovsky and colleagues 5 were the first to demonstrate a strong localized AcP positivity in cells of lymphocytic malignancies of T cell type. Since his initial report, Catovsky 6 and other investigators 19,20 ' 24 have obtained similar results. According to these ob- servers, AcP may be used as a simple and reliable marker for identifying T-lymphocytic proliferations. (Acid phosphatase positivity in an intensely localized form has been observed in a few cases of acute myeloblasts leukemia by Catovsky 8 and one of the authors, 14 an observation that does not invalidate the concept of AcP specificity for T cells in an otherwise proven neoplastic lymphoproliferative disease.) 318 PANGALIS, WALDMAN, AND RAPPAPORT However, in our study we failed to find any meaningful differences in either patterns or degrees of AcP positivity between B and T lymphocytes obtained from blood and tonsils of normal donors. Similar findings in blood samples have recently been reported by Wehinger and Mobius24 and confirmed in our laboratory by biochemical methods.15 In 1969, Tamaoki and Essner,22 in a study of AcP and j8G reactions on lymph-node frozen sections obtained from man and experimental animals, observed no enzymatic activity in the follicular and mantle lymphocytes, while AcP and BG activities were present in the paracortical lymphocytes. These findings were subsequently interpreted as evidence of a cytochemical difference between B (follicle and mantle) and T (paracortical) lymphocytes. Our results are consistent with Tamaoki and Essner's observations22 with respect to the BG findings, but not with respect to the AcP observations. The very low BG activity of tonsillar B lymphocytes in comparison with tonsillar T lymphocytes and blood B and T lymphocytes seems to indicate a difference between the B and T lymphocyte populations of the tonsils. This difference is probably not related to the B orT nature of the cells, but may be related to the state of the cell cycle of the follicular tonsillar B lymphocytes. The observation of a low BG activity in tonsillar B lymphocytes is of interest in view of the finding of a very low BG activity in the cells of lymph node imprints from diffuse non-Hodgkin's lymphomas."5 In summary, our findings revealed: 1. Very intense and localized a-NAE activity in the majority of the blood and tonsillar T lymphocytes, in contrast to very low a-NAE activity in the blood and tonsillar B lymphocytes; 2. Very low BG activity in tonsillar B lymphocytes, in contrast to a relatively normal BG activity in tonsillar T lymphocytes. The significance of these findings and their possible practical application to diagnostic hematology should be further investigated by studying malignant lymphocytic proliferations by both cytochemical and immunologic methods. Acknowledgments. Mrs. Aldana Martin and Mr. Michael D. Lockwood provided skillful technical assistance. References 1. Abdou NL, Alavi JB, Abdou Nl: Human bone marrow lymphocytes: B and T cell precursors and subpopulations. Blood 47:423-430, 1976 2. Barka T, Anderson PJ: Histochemical methods for acid phosphatase using hexazonium pararosanilin as coupler. J Histochem Cytochem 10:741-753. 1962 3. Bennett JM, Reed CE: Acute leukemia cytochemical profile: Diagnostic and clinical implications. Blood Cells 1:101-108, 1975 4. Brouet JC, Flandrin G, Sasportes M, et al: Chronic lymphocytic leukemia of T-cell origin. Immunological and clinical evaluation in eleven patients. Lancet 11:890-893, 1975 A.J.C.P. • March 1978 5. Catovsky D, Galetto J, Okos A. et al: Cytochemical profile of B and T leukaemic lymphocytes with special reference to acute lymphoblastic leukaemia. J Clin Pathol 27:767-771. 1974 6. Catovsky D: T-cell origin of acid-phosphatase-positivc lymphoblasts. Lancet 11:327-328, 1975 7. Catovsky D, Frisch B, Van Noorden S: B. T and "null" cell leukaemias. Electron cytochemistry and surface morphology. Blood Cells 1:115-124, 1975 8. Catovsky D: Classification of acute leukemia. Oral presentation at the Combined Clinical Staff Conference of the FrenchAmerican-British Cooperative Group. National Institutes of Health, Bethesda, Maryland, 1976 9. Flandrin G, Daniel M-Th: Cytochimie des esterases en hematologic. Pathol Biol 19:547-555, 1971 10 Flandrin G, Daniel MT: /3-Glueuronidase activity in Sezary cells. Scand J Haematol 12:23-31, 1974 11. Hayashi M, Nakajima Y, Fishman WH: The cytologic demonstration of /3-glucuronidase employing naphthol AS-B1 glucuronide and hexazonium pararosanilin; a preliminary report. J Histochem Cytochem 12:293-297, 1964 12. Li CY, Yam LT, Lam KW: Acid phosphatase isoenzyme in human leukocytes in normal and pathologic conditions. J Histochem Cytochem 18:473-481, 1970 13. Mueller J, Brun del Re G, Buerki H, et al: Nonspecific acid esterase activity: A criterion for differentiation of T and B lymphocytes in mouse lymph nodes. Eur J Immunol 5: 270-274, 1975 14. Pangalis GA: Personal observations 15. Pangalis GA, Kuhl W, Waldman SR, et al: Acid hydrolases in normal B and T blood lymphocytes. Acta Hematol (in press) 16. Pangalis GA, Yataganas X, Fessas Ph: /3-Glucuronidase activity on lymph node imprints of malignant lymphomas and chronic lymphocytic leukemia. J Clin Pathol 30:812-816, 1977 17. Pernis B, Forni L, Amante L: Immunoglobulin spots on the surface of rabbit lymphocytes. J Exp Med 132:1001-1018. 1970 18. Potvin C, Tarpley JL, Chretien B: Thymus-derived lymphocytes in patients with solid malignancies. Clin Immunol Immunopathol 3:476-481. 1975 19. Ritter J, Gaedicke G, Winkler K, et al: Possible T-cell origin of lymphoblasts in acid-phosphatase-positive acute lymphatic leukaemia. Lancet 11:75, 1975 20. Stein H, Petersen N, Gaedicke G, et al: Lymphoblastic lymphoma of convoluted or acid phosphatase type-A tumor of T precursor cells. Int J Cancer 17:292-295, 1976 21. Rozenszajn L, Marshak G, Efrati P: Acid phosphatase activity in normal human blood and bone marrow cells as demonstrated by the azo dye method. Acta Haematol 30:310-316. 1963 22. Tamaoki N, Essner E: Distribution of acid phosphatase, /3-glucuronidase and N-acetyl-/3-glucosaminidase activities in lymphocytes of lymphatic tissues of man and rodents. J Histochem Cytochem 17:238-243, 1969 23. Waldman SR, Roth JA, Silverstein M. et al: Effects on cancer patients of leukapheresis with the continuous-flow blood cell separator. 1. Hematologic and immunologic parameters in vivo. J Lab Clin Med 86:938-949. 1975 24. Wehinger N, Mobius W: Cytochemical studies on T and B lymphocytes and lymphoblasts with special reference to acid phosphatase. Acta Haematol 56:129-136, 1976 25. Winchester RJ, Fu SM, Hoffman T, et al: IgG on lymphocyte surfaces; technical problems and the significance of a third cell population. J Immunol 114:1210-1212, 1975 26. Yam LT, Li CY, Crosby WH: Cytochemical identification of monocytes and granulocytes. Am J Clin Pathol 55:283290, 1971 27. Yam LT, Mitus WJ: The lymphocyte /8-glucuronidase activity in lymphoproliferative disorders. Blood 31:480-489, 1968