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In 83 children with acute lymphoblastic leukaemia (ALL) the immunological phenotype of the lymphoblasts was determined using E rosetting, monoclonal anti-T cell sera, surface immunoglobulin staining and common ALL antiserum. The data were compared with acid alpha-naphthyl acetate esterase (ANAE) and acid phosphatase (AP) cytochemical data. The vast majority of T-ALL cells proved to be ANAE-negative (30/31) and AP-positive (29/31). Null-ALL was always AP-negative (12/12), the ANAE reaction was sometimes positive (4/12). In common ALL, findings ranged from negative to positive for both enzymes. Two cases of B-ALL were ANAE-negative and AP-negative. Enzyme cytochemical determinations gave thus preferential patterns, especially for T-ALL and null-ALL. In common ALL, considerable heterogeneity was found, which may be a reflection of differences in maturation between different cases of common ALL.

Focal cytoplasmic acetylesterase activity was sought in the malignant cells of 91 consecutive children with acute lymphoblastic leukaemia, of whom 10 had an anterior mediatstinal mass at diagnosis. This subgroup with thymic disease (TD) was characterized by hyperleucocytosis, the total leucocyte count being greater than 200 X 10(9)/1 in 6 patients. Furthermore, there was a significant association (P less than 0.025) between TD and the presence of leukaemic blast cells expressing a thymic phenotype, in the form of rosette formation with sheep erythrocytes. Focal cytoplasmic acetylesterase activity identified TD with a sensitivity of 20%, a specificity of 91%, a positive predictive value of 25%, a negative predictive value of 88% and a diagnostic accuracy of 81%. The demonstration of this feature by a simple cytochemical technique can be a useful component of the profile of investigations which are employed in the classification of acute lymphoblastic leukaemia.

Mononuclear cells (MNC) of 17 patients suffering from B chronic lymphocytic leukemia (B-CLL) were analysed by various immunological methods. The B cell nature of CLL cell was determined by classical tests (MRBC-rosette-test, immunofluorescence test for detection of membrane bound immunoglobulins). The cytochemical detection of the new T-cell marker dipeptidyl peptidase IV (DP IV) was found to be suitable for the characterization of B-CLL. The B-CLL cells showed granular pattern of alpha-naphthylacetate esterase (ANAE) reaction and binding of the monoclonal pan T antibody BL-T2. These non typical reactions for normal B lymphocytes can be used for differential diagnosis of B-CLL in combination with other reliable T cell markers. Avoiding the separation of T cells, the mixed rosette assay was used to enumerate Fc-IgG-receptor bearing T(TG) and non T cells. Both cell populations were found to be significantly elevated in MNC of B-CLL.

We describe an 8-year-old girl who presented a massive mediastinal tumour of thymic origin. Surgical excision was unsuccessful. Irradiation treatment (4,500 rad) together with intensive combination chemotherapy resulted in regression of the tumour. After three months, an increase of blasts was observed in the peripheral blood and bone marrow. The terminal phase was characterized by acute leukaemia, which did not respond to chemotherapy. Despite the thymic origin of this malignancy, the blast cells contained non-specific esterase activity as determined by naphthol-AS-acetate as substrate. The NaF-sensitive form of this enzyme is a characteristic of monocytes-macrophages. Other monocyte-macrophage markers were, however, negative. We conclude that the NaF-sensitive naphthol-AS-acetate esterase is not limited to tumours of the monocyte-macrophage system.

Tissue sections of lymph nodes, appendices and tonsils, together with smears of immunologically separated peripheral lymphoid cells from a B-CLL and lymphomatous cells from an immunocytoma were submitted to combined enzyme cytochemical investigations with acid alpha-naphthyl acetate esterase (ANAE), beta-glucuronidase (B-G), acid phosphatase (AcPh), adenosine triphosphatase (ATPase), a,d 5'nucleotidase (5'N). T-cells were Acph+, ATPase- and 5'N-. The vast majority of T- and B-cells displayed ANAE and B-G activities with two distinct staining patterns (T-like and B-like pattern). A high proportion of lymphoid cells in the germinal centre (G.C.) and the vast majority of lymphoid cells in the mantle-zone (M.Z.) were shown to belong to B-cell system because of the expression of ATPase and 5'N in their membranes. Some lymphoid cells positive for ANAE and B-G with a B-like pattern and for AcPh were recognizable in the G.C. In the M.Z. only a few lymphoid cells being ANAE+, with a T-like pattern, and AcPh+ were shown to belong to the T-cell system. In contrast, in this zone a high proportion of small lymphoid cells (64% +/- 10%) showed ANAE activity, mostly with B-like pattern. Therefore, these findings indicate that in the M.Z. a high proportion of B-cells ATPase+ and 5'N+ also display ANAE activity. By comparison of the results obtained from lymphoid tissue sections, B-CLL and immunocytoma cell suspensions and normal circulating lymphocytes we can conclude that B-ANAE-positive cells of the M.Z. do not usually appear in the peripheral blood. They circulate in large numbers only in some pathological conditions (like our reported B-CLL). Therefore, B-ANAE-positive lymphoid cells of the mantle, with a B-like staining pattern, include a wide range of subsets which exclude large lymphoid cells and plasma cells.

Previous studies have shown that a distinctive pattern of acid alpha-naphthyl acetate esterase (ANAE) activity (focal reaction product) characterizes normal human peripheral blood and tissue T lymphocytes but is absent from thymocytes and certain mitogen-stimulated T-cell blasts. In the present study mononuclear cell suspensions prepared from the peripheral blood and tissue specimens of 35 patients with lymphoid malignancies were simultaneously analyzed for surface immunoglobulin, sheep erythrocyte rosette formation, Ia antigens, and ANAE activity. The neoplastic cells from 16 patients with Ia+ SIg+ E- (B cell) malignancies, 4 patients with Ia+ SIg- E- (non-B, non-T) acute lymphoblastic leukemia, and 3 patients with Ia- SIg- E- (null cell) malignancies failed to exhibit ANAE activity. The neoplastic cells from 5 patients with Ia- SIg- E+ (T cell-derived) malignancies, including three cutaneous lymphomas, displayed characteristic T-pattern positivity, and in each case the percentage of E+ and ANAE+ cells was comparable. The neoplastic cells from 4 patients with Ia- SIg- E+ (T cell-derived) acute lymphoblastic leukemia were ANAE-. The expression of ANAE activity in T cell-derived malignancies may parallel its expression in the stages of normal T-cell differentiation and may prove to be a useful marker with which to sort out T-cell phenotypes.

2 cases of T-cell prolymphocytic leukaemia (T-PLL) were investigated for their reactivity with a series of monoclonal antibodies (MoAbs) as well as for the cytochemical expression and functional activity of the pathological cells. Both patients showed morphological (large cells with abundant cytoplasm and eccentric and irregularly shaped nucleus with large and prominent nucleoli) and clinical (high leucocyte count and splenomegaly) features typical of T-PLL. The cells from 1 patient expressed a helper/inducer phenotype (T4+, T8-) and were reactive with the anti-Tac (interleukin-2 receptor) MoAb, while the other case co-expressed both the T4 and the T8 antigens. The response to phytohaemagglutinin and the natural killer activity (assessed by 51chromium release) were significantly reduced in both cases, while the helper capacity, tested in a pokeweed mitogen-driven system, was maintained only in the 1st case. This latter case which expressed a more mature phenotype (T4+, T8-) responded well to chemotherapy.

A 76-year-old woman with acute leukemia responded incompletely to prednisone and vincristine. Cytochemistry of the blast cells demonstrated only focal alpha-naphthyl acetate and alpha-naphthyl butyrate esterase activity and focal coarse granular beta-glucuronidase activity, a pattern usually associated with acute lymphocytic leukemia. Electron microscopy demonstrated primitive cells with features usually associated with promonocyte differentiation including prominent parallel arrays of microfilaments and nucleoli of the nucleolonema type. In Wright-stained films, blasts contained oval, pale-blue, yellow tinged cytoplasmic inclusions about 2 to 4 micrometers in diameter which did not stain with any of the cytochemical methods employed. With electron microscopy, these inclusions were nonmembrane bound structures, varying from amorphous electron dense stippling to irregularly arranged short segments of filaments to prominent parallel arrays of filaments. We propose that the inclusions may have arisen from the parallel arrays of microfilaments and are nonspecific.

The acid phosphatase (AP), beta-glucuronidase (BG), and alpha-naphthyl-acetate esterase (ANAE) distribution and the morphology of stage 0 B-CLL lymphocytes were studied. The results were compared with the same hydrolase equipment and morphology of normal B-cell populations showing the B-CLL-like phenotype, and thus regardable as the possible counterpart of leukaemic cells. The functional and structural characters of the former were strikingly different from those of the latter. In fact the normal B-cell population with B-CLL-like phenotype showed an homogeneous enzyme pattern with predominant ANAE and a strictly lymphocytic cell morphology. In contrast, the leukaemic cells showed various and unrelated morphological and cytochemical features, thus forming apparent subgroups of B-CLL. The development of an irregular "switch on" mechanism in the blocked malignant cell clone, may account for these functional and structural maturation discrepancies. Moreover, the fact that in the leukaemic cells ANAE activity could be demonstrated only after the appearance of the other hydrolases, makes it a marker of functionally differentiated B lymphocytes.

Four acid hydrolases, acid phosphatase (AP), alpha-naphthyl acetate acid esterase (ANAE), beta-glucuronidase (BG) and N-acetyl-beta-glucosaminidase (NABG) were determined cytochemically in B-chronic lymphocytic leukaemia (B-CLL) cells exposed in vitro to the tumor promoter 12-0-tetradecanoyl phorbol 13 acetate (TPA). TPA, which has been previously shown to induce B-CLL cells to mature towards plasmacytoid cells, results in the progressive expression of the enzymes tested in the cytoplasm of malignant cells, in particular AP and ANAE. Furthermore, the sensitivity to inhibitors and the pattern of reactivity of ANAE provide evidence for an enzyme subtype normally restricted to plasma cells. Thus, acid hydrolases--some of which showing plasma cell type of activity--are expressed during B-CLL cells differentiation induced in vitro. These results confirm the value of cytochemistry in subtyping B-cell malignancies.

The pattern of staining for ANAE in lymphocytes, plasmocytes, monocytes and leukemic cells has been studied and the effect of NaF and E 600 on this staining reaction has been investigated. In lymphocytes the transition occasionally occurring between a dot-like (T type) and granular positivity may cause difficulties when using this reaction as a marker of resting T lymphocytes. In plasmocytes as well as in myeloma cells a number of coarse granules was present in most cells and in some cases even large blocks of the reaction product occurred. NaF and E 600 inhibits already in lower concentration (2 mM and 10 micronM respectively) the strong diffuse reaction in monocytes, the effect on other cells being negligible. Using higher concentrations the inhibitory effect of NaF (20 mM) was more pronounced in plasmocytes in comparison with T lymphocytes which on the other hand, were, in general more sensitive to E 600 (10 mM). Partially diffuse or dense granular pattern of ANAE was found in a number of hairy cells. "Lymphoblastic" leukemias in children most frequently displayed negative or fine granular reaction in a varying number of blasts and similar findings were noted in the majority of cytochemically undifferentiated leukemias in adults. In some of them, however, a similar pattern of ANAE as in myeloblastic leukemias with dispersed or partial diffuse positivity was observed. The significance of these findings is briefly discussed.

A diagnosis of prolymphocytic leukemia was made from the blood and bone marrow of a 50 year old man. The neoplastic cells were studied by use of light and electron microscopy. Neoplastic cells were focally positive for acid phosphatase and alpha naphthyl acetate esterase. In addition, neoplastic cells formed rosettes with sheep erythrocytes and reacted with Leu-1 and Leu-2a but not Leu-3a antisera. No terminal deoxynucleotidyl transferase (TdT) activity was noted in these cells. It is concluded that these neoplastic cells were phenotypically mature suppressor T lymphocytes. Furthermore, T and B cell prolymphocytic leukemias were compared according to clinico-pathological, cytochemical, ultrastructural, and immunological findings derived from our review of the current literature.