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Four cell lines were established from peripheral blood of patients with leukemia. All lines express the Epstein--Barr virus nuclear antigen (EBNA) and therefore should be classified as lymphoblastoid cell lines. However, one of the lines UHKT-5 established from a patient with acute myelomonocytic leukemia has some features not typical for lymphoblastoid cell lines. The cells resemble to macrophages with the phagocytic ability for yeasts, strong alpha-naphthylacetate esterase positivity in phagocytizing cells and unusually long villi. Another line UHKT-7 established from a patient with hairy cell leukemia expresses the same isotype of membrane immunoglobulins as the original hairy cells.

The numbers of alpha-naphthyl acetate esterase (ANAE)-containing cells (other than T lymphocytes) in non-Hodgkin's lymphomas (NHL) and reactive lymph nodes have been counted, using the Reichert-Jung (Kontron) MOP-AMO3 user-controlled image analyzer. Twenty specimens of NHL and ten reactive nodes were examined. Cells were demonstrated by their content of acid alpha-naphthyl acetate esterase (ANAE) in fixed frozen sections. It was found that lymphomas of high-grade malignancy contained much larger numbers of ANAE-positive cells (10.8-20.5%) than those of low-grade malignancy (1.4-4.1%). The number of ANAE-positive cells (1.4-3.2%) in reactive lymph nodes was similar to that in low-grade NHL nodes.

A comparative study of alpha-naphthyl (nonspecific) esterase (ANAE), acid phosphatase (AcP), and beta-glucuronidase (beta-GLUS) in the blood lymphocytes was carried out on 26 healthy pregnant women (7 to 27 weeks' amenorrhea) and on 32 normal nonpregnant women, all 58 of whom were 19 to 34 years old. In pregnant women, we found a significantly lower percentage of ANAE-positive lymphocytes (after 11 weeks' amenorrhea), a strong reduction in AcP, and a slight decrease in beta-GLUS. The modified enzyme levels of circulating lymphocytes in the early stages of pregnancy seem to be related to the depressed cell-mediated immunity.

The alpha-naphthyl acetate esterase (ANEA) is a histochemical marker for human T lymphocytes in cell smears and frozen tissue sections. We have now applied the ANAE method to paraffin-embedded tissue sections. We first demonstrated with cytocentrifuged cell smears of blood leukocytes that the ANAE activity is preserved upon prolonged storage in formol calcium, Holt's buffer, acetone, xylene, and heat. When the tissue sections were similarly processed and embedded in paraffin, the ANAE positive (T) lymphocytes were identified by their distinct display of one or more reddish-brown reaction dots in the cell cytoplasm. ANAE positive mononuclear phagocytes were easioy distinguished from the T lymphocytes by their diffuse, sodium fluoride-sensitive pancytoplasmic reaction. The extension of the ANAE method to paraffin-embedded tissue sections with superior morphological integrity, makes it possible to apply it in practical biopsy pathology.

We have previously shown that non-proliferating human T- but not B-lymphocytes contain demonstrable amounts of acid alpha-naphthyl acetate esterase (ANAE). The usefulness of this histochemical marker for the diagnosis and classification of malignant lymphoid tumors was investigated by use of a panel of established normal and malignant human haematopoietic cell lines and fresh biopsy cells from malignant lymphomas and myelomas. The results showed that not only the T-cell derived acute leukaemia lines, but also histiocytic lymphoma and myeloma lines and some of the lymphoma (Burkitt and lymphocytic) and non-neoplastic lymphoblastoid cell lines with B-cell surface markers expressed strong ANAE reactivity. Some but not all of the immunoglobulin producing myeloma and lymphocytic lymphoma biopsies were ANAE-positive. Inhibition experiments with sodium fluoride and E-600 demonstrated that although the T-lymphocyte specific esterase is predominantly of 'A'-type, the malignant lines contain also non-specific 'B' esterase and pseudocholinesterase. As the presence of the various esterases did not demonstrate any specific distribution pattern among he haematopoietic cell lines of different origin, we concluded that the ANAE marker is no longer T-specific when malignant lymphoid cells are considered, and that the usefulness of this marker in routine diagnostic work therefore is limited.

Human monocytes contain a series of alpha-naphthyl acetate (alpha NA) esterases which are not present in other blood cells and which can be specifically inhibited by bis(4-nitrophenyl)-phosphate (BNPP). This inhibitor is non-toxic at the concentration used and thus enabled studies on the possible significance of this enzyme towards various monocyte functions. BNPP has no noticeable influence on adhesion and spreading of monocytes on glass surfaces, nor does it inhibit the phagocytosis of IgG-coated latex particles. BNPP does, however, diminish the spontaneous cytotoxicity of freshly isolated monocytes towards the erythroleukaemic cell line K562. In the single cell assay in agarose, BNPP treatment of monocytes leads to a decrease in the number of lytic and non-lytic effector-target cell conjugates. In contrast, the antibody-dependent cell-mediated cytotoxicity (ADCC) of monocytes as well as the natural cytotoxicity of lymphocytes towards K562 cells are not influenced by BNPP. The present investigations show that monocyte specific alpha NA esterases are involved in the spontaneous cytotoxicity of monocytes toward tumour cells.

The localization of alpha-naphthyl acid esterase (ANAE) activity in T lymphocytes with receptors for IgM (T(M) cells) have been studied at the electron microscope. The electron-opaque product of the cytochemical reaction was detected around, but never inside, single (or groups of) vesicles, which suggested a possible membrane localization of the enzyme activity. These same vesicles were found to contain acid phosphatase by both light- and electron-microscopic examination and were bound by unit membranes; this data indicates that they likely represent primary lysosomes. The presence of such lysosomes in a restricted paranuclear area is a distinctive feature of T(M) cells.

The clinical, morphologic, and cytochemical findings of 7 patients with malignant histiocytosis are presented in this study. The diagnoses were confirmed by the use of electron microscopy and cytochemistry (acid phosphatase and naphthol-AS-acetate-esterase). Two different types of malignant histiocytes were identified by the differentiation and development of cell organells. The relation of malignant histiocytosis to monocytic leukemia and to normal histiocytes (and their precursors) is discussed. And finally, there are statements on the origin and peculiarities of malignant histiocytes.

A modified technique for demonstration of acid alpha-naphthylacetate esterase was examined with regard to specificity and application on human T-lymphocytes in peripheral blood and pleural fluids of different genesis. T-lymphocytes of healthy donors showed only in 60% positive findings of acid alpha-naphthylacetate esterase. Positive reactions appear as dot-like reaction products in cytoplasm of lymphocytes. The application of this method on pleural fluids is possible, without technical problems. We found differences in the percentage of positive-reacting lymphocytes in pleural fluids between tuberculosis (= 78% T-cell esterase positive lymphocytes) and tumors (= 65% T-cell esterase positive lymphocytes). Mesotheliomas exhibit very low positive findings (= 32% T-cell esterase positive lymphocytes) in contrast to carcinomas. In this way, acid alpha-naphthylacetate esterase may be helpful in some cases as a diagnostic aid.

17-Chloroacetylajmaline and 17-acetylajmaline are reported to have in vivo antiarrhythmic activity and are metabolized by hydrolysis. Since the hydrolysis product, ajmaline, may be the actual antiarrhythmic agent, the hydrolysis of these derivatives by various tissues of the guinea pig, rat, and mouse was determined in vitro by a titrimetric method and compared to hydrolysis by alpha-naphthylacetate. The heart is the most active tissue in the guinea pig for hydrolyzing 17-chloroacetylajmaline. The hydrolyzing activity is greater in the guinea pig than in rat or mouse heart, corresponding with the more significant pharmacological activity in the guinea pig. 17-Chloroacetylajmaline has a significantly lower Km value than 17-acetylajmaline, which is in agreement with the in vivo activity.

Alpha-Naphthyl acetate esterase (ANAE) and fluoride resistant alpha-naphthyl acetate esterase (FRANAE) have been compared as histochemical methods to identify T lymphocytes in sections of normal and pathological human lymphoid tissues. In addition, the FRANAE method was combined with alkaline phosphatase (ALP) in order to simultaneously evaluate the relationship between T lymphocytes and fibroblastic reticular cells (ALP) positive). The "dot like" esterase positivity of T lymphocyte was better evaluated by using FRANAE when compared to ANAE because of fluoride inhibitor of the strong esterase activity of dendritic cells and most macrophages. The combined ALP-FRANAE method clearly demonstrated a large number of fibroblastic reticular cells within the T-areas in various normal and pathological tissues such as hyperplastic lymph nodes and especially in the lymph nodes and spleens from patients with Hodgkin's disease.

Human peripheral blood mononuclear cells were stimulated with pokeweed mitogen (PWM) to study the role of mononuclear phagocytic cells (MNP) in lymphocyte proliferation. MNP were identified by cytoplasmic alpha-naphthyl acetate esterase, by the capacity of phagocytosis and by lysozyme synthesis. It appeared that after 3 days of stimulation with PWM all MNP disappeared from the cultures and remained absent during prolonged culture in mitogen-free medium. In non-stimulated cultures MNP remained. The disappearance of MNP from cell cultures was caused by a lymphocyte-derived factor, which was transferable by cell-free supernatants of stimulated mononuclear cells. From experiments in which cultures were treated with different concentations of PWM and from pre-culture experiments, it could be shown that in vitro lymphocyte proliferation required both non-stimulated lymphocytes and freshly prepared MNP. In addition, the decreasing concentration of PWM as stimulating agent, resulted in a decreasing proliferation of lymphocytes, which was inversely proportional to the presence of MNP.

Acid alpha-naphthyl acetate esterase (ANAE) activity was investigated in nucleated red cells obtained from nine human fetal livers at 9-24 weeks of gestation. A strong ANAE activity was demonstrated in fetal liver erythroblasts with a staining pattern of a perinuclear ring. Occasionally some polychromatophilic normoblasts expressed a focal ANAE activity usually observed in human T-lymphocytes. Some 50-70% of erythroblasts in human fetal livers showed a ring-like staining pattern. A slight decrease of ANAE positivity was observed in cells of fetal livers after 17 weeks of gestation.

The authors report their experience of a histo-enzymatic method, using Naphtol AS-D Chloroacetate as a substrate, for the specific demonstration of elements of the granulocyte series, in histological sections, after formol fixation and mounting in paraffin wax. This simple and reliable staining technique offers the possibility of very rapid diagnostic orientation in haematological conditions.

The Vickers M86 integrating microdensitometer has been used to quantify the cytochemical reaction for acid naphthol AS-BI phosphatase activity in isolated, rat liver parenchymal cells. Data are presented which validate the method. The use of this method together with that of the Feulgen reaction to estimate nuclear ploidy value in the same cell, has shown that there is an increase in acid phosphatase activity of up to 100% when the euploidy value of the liver cell doubles. It has been further shown that 70--80% of this enzyme activity is ouabain-sensitive, regardless of the euploidy value. The data may be interpreted to indicate that the extra gene copies of the polyploid cells are operative.

The histrochemistry of the adrenal glands was studied in four adult male marmosets (two Callithrix jacchus and two Callithrix penicillata). It was impossible to demonstrate any reactivity to UDPG-GT, ADH, alanyl aminopeptidase, leucine aminopeptidase, xilitol (NAD-dependent) dehydrogenase, beta-glucuronidase and aryl-sulfatase in these glands. Total phosphorylase was found in scattered cells of the glomerulosa and adjacent outer fasciculata of one C. penicillata. The dehydrogenases (LDH, G-6-PDH,6-PGDH, NADPH2-TR,ICDH,SDH,NADH2-TR, alpha-GPDH, beta-OHBDH) as well as the hydrolases (except alkaline phosphatase, ATPase, and acetylcholinesterase) showed a stonger reactivity in the cortical part. Some hydrolases (naphthol acetate esterase, acid phosphatase) and cytochrome oxidase were less reactive in the zona glomerulosa, where the dehydrogenases were more abundant. The outer fasciculata and the reticularis also showed a strong dehydrogenase reactivity.

The separation, characterization and functional assay of the inflammatory infiltrate present in the site of the lesion has been useful in the study of many diseases. Histochemical techniques for esterase and acid phosphatase, as well as the Phagocytose test and the Giemsa staining were applied to the study of the spleen-cell population of ten mice. A good characterization of the components of the Phagocytic Mononuclear System and the identification and quantification of the total cell population were obtained.

Human erythrocytes contain 7 electrophoretically different alpha-naphthyl acetate esterases. Their solubility properties indicate that 5 are present in the erythrocyte cytoplasm and 2 are tightly bound to the stroma; the stroma-bound esterases can be inhibited by 10(-5) M physostigmine. No additional physostigmine-sensitive enzymes were detectable in Plasmodium falciparum-infected cells. Addition of physostigmine to malaria parasites in culture killed the parasites rapidly.

Natural killer (NK) cells have been obtained from mouse spleens and grown in vitro. These cells: retained cytotoxicity against YAC targets; and were homogeneous as judged by morphology and surface markers. The alpha-naphthol acetate esterases (ANAE) and diisopropyl-fluorophosphate (DFP)-binding proteins of NK cells, YAC cells and NK-YAC conjugates have been examined. Ultrastructural cytochemistry indicates that NK cells have two types of ANAE: an enzyme primarily associated with the granule externum and an activatable ANAE associated with the granule internum. Both of these activities are inhibited by DFP. The activatable ANAE appears during incubation of NK cells with YAC cells. DFP-binding proteins were examined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and autofluorography. NK cells and YAC cells have major DFP-binding proteins of 35 X 10(3) and 20 X 10(3) Mr, respectively. NK-YAC conjugates had a new band at 55 X 10(3) Mr. This new protein may be identical to the activatable ANAE described above. Our studies provide evidence for an NK cell esterase that becomes activated when incubated in the presence of tumour cells. This esterase may be related to the cytolytic mechanism.

Monocytopoiesis and blood monocytes were investigated in nine patients with active tuberculosis and in six patients with active sarcoidosis in order to obtain information on monocyte consumption in these two types of granuloma. All patients with tuberculosis demonstrated a marked increase in proliferation activity of monocytopoiesis and premature monocyte marrow release. These changes indicate a high monocyte consumption which probably is caused by a high macrophage death rate due to the high macrophage-toxicity of tubercle bacilli. Thus, tuberculous lesions are an example of a "high turnover granuloma". In sarcoidosis monocytopoiesis showed no significant deviations from the normal. This indicates a low macrophage turnover or "low turnover granuloma". Thus, any hypothetical agent assumed to be involved in the pathogenesis of sarcoidosis would have to possess low macrophage-toxicity.

Alpha-naphthyl acetate esterase (ANAE) activity in alveolar macrophages was demonstrated in air-dried smears from samples of 82 sputa, 47 bronchial secretions and 14 bronchial lavages from 113 patients. Enzyme activity was estimated by a semiquantitative scoring method. There was a 7.4% loss of activity after 24 hours of storing the unfixed material, increasing to 14.4% after three days, while storing the air-dried smears for up to four weeks did not change the ANAE activity. The mean cellular esterase activity was correlated to the clinical and cytologic findings. A stringent correlation could be found for patients smoking more than 30 cigarettes a day; they had a 17% increase in activity as compared to nonsmokers. In patients with bronchial asthma, the activity was 18% higher than the total mean. In three patients with pulmonary embolisms, the ANAE activity was also increased. Treatment with a combination of cytostatics and a corticoid caused a severe decrease. No correlation could be found to age, sex, inflammation or malignant or cardiac diseases. These findings indicate that the application of the ANAE reaction to routine cytologic specimens can contribute to the functional characterization of human alveolar macrophages.

Utilizing the acid alpha-naphthyl acetate esterase histochemical staining technique, cells with established T-cell (thymus-derived) staining characteristics could be consistently demonstrated in the periarteriolar lymphatic sheaths of the spleen and the diffusely infiltrated area of the cloacal bursa. The same areas failed to stain positively in significant numbers following a preincubation period with a specific inhibitor for carboxyl esterase. Lymphoid cells in the thymus, the splenic follicles, and bursal follicles were mostly negative.

Monoclonal antibodies reactive with T cells, T cell subsets, B cells, monocytes, and natural killer cells were used to characterize the nature of mucosal lymphocytes in the human small intestine by application of the immunoperoxidase technique to tissue sections for light and electron microscopic examination. In addition, for comparison, peripheral blood mononuclear cells (PBL) were studied by immunoelectron microscopy. Most of the intraepithelial lymphocytes (IEL) were T cells (Leu-1+, T3+) and expressed the phenotype associated with cytotoxic/suppressor T cells (Leu-2a+, T8+). In contrast, a majority of T lymphocytes in the lamina propria expressed the phenotype associated with helper/inducer T cells (Leu-3a+, T4+). These observations confirm and extend the findings previously reported. In addition, a small number of cells in the lamina propria with the ultrastructural features of macrophages were found to react with anti-Leu-3a and anti-T4 antibodies. Although many IEL contained cytoplasmic granules and had ultrastructural features similar to those of circulating granular lymphocytes, none of these cells reacted with anti-Leu-7 (HNK-1), anti-T10, or anti-M1 antibodies. This suggests that IEL may not be related to circulating large granular lymphocytes, which are Leu-7+, T10+, M1+ and are associated with natural killer activity. Not only Leu-7+ PBL, but T8+, T4+, or T3+ mucosal lymphocytes or PBL also may contain cytoplasmic granules. Therefore, the cytoplasmic granules are not restricted to one cell type, in particular, to Leu-7+ cells.

We examined a series of hepatocellular neoplasms, including 4 adenomas, 7 hepatoblastomas, and 18 hepatocellular carcinomas (HCC) with enzyme histochemistry in plastic-embedded sections. Our most striking observation was that there was a distinct difference in the staining pattern for alkaline phosphatase (Alk0) in benign and malignant tumors. Non-neoplastic controls (normal liver, reactive lesions) and benign neoplasms showed a distinctive canalicular pattern of staining with Alk0. Malignant neoplasms, however, showed a virtual absence of Alk0 staining; 6 of 7 hepatoblastomas and 17 of 18 HCCs were practically devoid of staining, while the two positive cases showed a pattern easily discernible from normal. The sensitivity of the observed Alk0 staining pattern in detecting malignant hepatocellular neoplasms was 92% and the specificity was 100%. Cytoplasmic gamma-glutamyl-transferase (GGT) was present in a minority of HCCs, but faint staining was also seen in normal liver or in adenomas. It appears that these nonmorphologic techniques may aid the surgical pathologist in the differential diagnosis of primary hepatocellular neoplasms.

A series of monoclonal antibodies reactive with normal myeloid cells at different stages of differentiation (anti-MY4, -MY7, -MY8, -Mo1, -Ia) were used to characterize the leukemic cells of 70 patients with acute myeloblastic leukemia (AML). Sixty-two of the leukemias expressed a phenotype corresponding to a recognizable immature normal myeloid cell. These 62 cases could be divided into 4 phenotype groups, corresponding approximately to the normal CFU-C (group I, 21%), myeloblast (group II, 26%), promyelocyte (group III, 8%), and promonocyte (group IV, 45%). Morphological subtyping of these leukemias tended to agree with the immunologic phenotype, particularly with more "differentiated" morphological subtypes, such as acute monocytic leukemia or acute promyelocytic leukemia. However, each phenotype group contained more than one morphological type of AML, indicating that the level of differentiation of the surface membrane of AML cells may not always be concordant with morphology. The phenotype groups were also analyzed with respect to cytochemical staining patterns, age, the presence of Auer rods, and complete remission rates. Statistically significant differences among the groups were noted in the distribution of myeloperoxidase staining, nonspecific esterase staining, and Auer rods. The complete remission rates varied from 60% (groups III and IV) to 88% (group II). These results suggest that surface marker analysis in AML may be used as a highly reproducible classification system that will provide additional information about the leukemic cells in conjunction with morphological analysis.

Acid alpha-naphthyl acetate esterase (ANAE) activity was assayed in cell homogenates and in intact cells by an endpoint colorimetric method, in which sodium dodecyl sulfate was used to stop the reaction. Each method of cell disruption and enzyme solubilization tested here caused a partial loss of the ANAE activity in lymphocyte preparations. The majority of the ANAE activity in lymphocytes was found to be membrane bound. The ANAE activity in thymocytes was over two times lower than that obtained for lymph node and spleen lymphocytes. Macrophages were found to contain about 18 times higher ANAE activity than mature lymphocytes.

A histochemical investigation of 16 sites of post-mortem oral mucosa was carried out in an attempt to define any organized lymphoid tissue in the mouth. Whereas lymphoid tissue was found, as expected, in the palatine and lingual tonsils, only 1 out of 240 specimens revealed lymphoid aggregations at other mucosal sites. However, mononuclear cells were scattered in the lamina propria of the oral mucosa and this varied from 53 cells/mm2 in the fetus to 83 cells/mm2 in old age. Non-keratinized oral mucosa showed a significantly higher number of mononuclear cells than keratinized mucosa. Up to 53 per cent of the cells stained with acid alpha naphthyl acetate esterase (ANAE), consistent with T cells, and up to 25 per cent gave a staining reaction consistent with monocytes. Some of the ANAE-negative cells were B lymphocytes. The presence of T cells was also examined by using the acid phosphatase reaction. It is suggested that, with the exception of the palatine and lingual tonsils, organized lymphoid tissue is not normally found in the rest of the mouth. However, the presence of scattered mononuclear cells at all ages and in all the sites of the oral mucosa examined suggests that these cells may provide a local surveillance mechanism.

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.

Epithelial cells filled with lymphocytes (nurse cells, NC), recently described in mouse, rat and human thymus, have been interpreted as mediators of direct contact ('stromal') induced thymocyte maturation. We describe analogous NC in trypsin-dissociated human adenoids and tonsils. NC from these organs show morphological characteristics analogous to those of thymic NC: they appear as large (diameter 30-35 micrometers) elements, containing peripherally situated tonofilament bundles, electrodense mitochondria and some vacuoles. Each NC contains 5-30 intact lymphoid cells, some of which appear in the activated state. NC show neither phagocytic ability, nor ANAE and peroxidase cytochemical reactions. The majority of NC from adenoids and tonsils react with the monoclonal antibody (McAb) OKIa1 (DR w framework) as those from thymus, and 40% of them bind fluorescein-conjugated peanut agglutinin. Some of them also react with the McAb OKT3 (pan-T), OKT9 (transferrin receptors) and OKT10 (immature hematopoietic cells). The presence of NC in adenoids and tonsils suggests that these organs may be involved in some stages of lymphocyte maturation requiring intimate contact with epithelial cells.

The pattern of alpha-naphthyl-acetate esterase (ANAE) activity in stromal and lymphatic cells has been examined in lymphatic organs from normal and hydrocortisone (HC)-treated mice. The distribution of ANAE-positive lymphocytes as well as stromal cells in peripheral lymphatic organs was different in T- and B-dependent regions, while in the thymus there was the difference between cortex and medulla. The HC application evoked a marked changes in the pattern of ANAE-positive cells distribution of the thymus, producing only a slight ones in the remaining organs. The influence of HC on the ANAE reaction intensity and distribution in the examined tissue sections is discussed.

The relationship between cell features by light microscopy and therapeutic outcome of 72 patients with acute myeloblastic leukemia (FAB M1) were investigated. The patients were divided into two groups according to myeloperoxidase-(MPO) positive percentage of blast cells, namely a low (less than 50%) and high (greater than 50%) MPO group. No remarkable morphological difference of blast cells between these two groups was observed. The 38 patients with low MPO showed a significantly lower complete response rate (CR) (52.6%) than the 34 patients with high MPO (85.3%) (p = 0.003) that included 10 CAE-positive patients and 2 ANAE-positive CAE-negative patients. The low MPO group included 5 CAE-positive patients with granulocyte dysplasia and 7 additional patients with alpha naphthyl acetate esterase (ANAE) positivity who were chloroacetate esterase (CAE) negative. Low positivity of blast cells may be due to a defective enzyme expression in the former but also may be a reflection of 'monocytic' aberrant expression in the latter. The low MPO group in M1 with this heterogeneous population is less likely to achieve CR with chemotherapy, while the high MPO group with a higher CR rate suggests a more pure entity within the myeloblastic leukemias referred to as M1. After further refinement by eliminating 24 cases that were 'esterase positive' (ANAE and/or CAE), the results were still the same, suggesting that the more immature blast populations were in the low MPO group.

Certain tissues, such as the spleen, are rich sources of mononuclear phagocytes (MP); however, separating the phagocytes from tissues and removing the contaminating cells have been difficult. We report here a method for the extraction and purification of human splenic MP that employs gentle homogenization of splenic fragments with a Tenbroeck tissue homogenizer, controlled digestion with purified collagenase to free MP from splenic stroma, incubation with DNase to dissociate cell clumps and purification by countercurrent centrifugal elutriation (CCE). With homogenization and enzymatic digestion most of the splenic nonspecific-esterase-positive cells are freed into suspension as determined by morphometric analysis of 2 micron sections from plastic embedded spleen stained for alpha-naphthyl butyrate esterase (ANB). Overall cell recovery after homogenization and enzyme treatment is 56 +/- 7%; no selective cell loss occurs as determined by differential cell counts at each purification step. CCE of up to 3 X 10(9) treated spleen cells results in recovery of 63 +/- 6% of the elutriated cells and separates nearly 50% of the recovered MP into enriched fractions. These MP are morphologically intact as determined by light and electron microscopy and are actively phagocytic. Highly purified (96%) autologous splenic lymphocytes are a useful by-product of this separation technique.

The use of unfixed and undecalcified cryostat sections of mouse knee joints is described for the study of enzyme histochemical reactions. Non-inflamed knee joints and knee joints of mice with antigen induced arthritis have been used. Joints were embedded in gelatin and subsequently cut at low speed with a motor-driven cryostat fitted with a tungsten carbide knife at an obtuse angle (10 degrees). The sections were attached to transparent tape to keep the integrity of the tissue intact. The following histochemical reactions were carried out successfully: the tetrazolium salt reaction for dehydrogenase and reductase activity, the post-azo-coupling method for acid phosphatase and cathepsin B activity and the simultaneous azo-coupling method for esterase activity. In all cases the morphology and integrity of the sections were well kept and serial sections were obtained without any difficulty. Nonspecific staining of the tape did not occur. The localization of the final reaction product was meeting criteria for specific and precise histochemical methods with the exception of the metal salt method because of nonspecific staining of undecalcified bone. Cytophotometry of the final reaction product appeared to be reproducible and valid as demonstrated by reaction for glucose-6-phosphate dehydrogenase activity in synoviocytes from knee joints with induced arthritis. End point measurements as well as kinetic measurements of the formazan production were performed and linear relationships were found between the specific formazan formation and section thickness or incubation time, respectively. It is concluded that cryostat sections attached to transparent tape are an excellent tool for the study of the metabolism in tissues adjacent to bone matrix.(ABSTRACT TRUNCATED AT 250 WORDS)

Parotid adenolymphoma is composed of two histologic components, epithelial and lymphoid. Although some theories regarding the histogenesis of this tumor have long been disputed, there have been no definite conclusions. The purpose of this study was to clarify the origin of the epithelial components of this tumor using histochemical and immunopathological techniques, electron microscopy and a survey of HE-stained tumor sections. The results obtained indicated that the functions of the epithelial components were similar to those of the striated duct of the normal parotid gland, and morphological studies showed that the origin of the epithelial components may arise from parotid ductal inclusion in the lymphnodes in or around the parotid gland.

The simultaneous autoradiography and the appearance of acid alpha-naphthylacetat esterase as a cytochemical T cell marker show that only in few T cells of the tonsil a DNA-synthesis takes place. Their labelling index is lower than a power of ten compared to B cells.

Differentiation of leukemic cells in vitro is characterized by the sequential appearance of morphological, functional, and biochemical markers of maturation. The interaction of insulin with its receptor may be a regulator of growth and differentiation of leukemic cells. Human promyelocytic leukemia cells (HL-60) demonstrate specific reversible insulin binding consistent with properties of human insulin receptor. HL-60 cells treated with 500 microM N6,O2-dibutyryl adenosine 3',5'-cyclic monophosphate, 1 microM 1 alpha, 25-dihydroxyvitamin D3, or 41 nM phorbol-12-myristate-13-acetate expressed monocytic markers of differentiation and an increase in insulin receptor expression. The change in insulin receptor expression with 1 microM 1 alpha, 25-dihydroxyvitamin D3 and N6,O2-dibutyryl adenosine 3',5'-cyclic monophosphate induction was further characterized by Scatchard analysis. High affinity binding (Kd) constant was not altered, and the change in binding was attributed to receptor number. Commitment to increased insulin receptor expression was demonstrated after 1-h exposure to 1 microM 1 alpha, 25-dihydroxyvitamin D3. Agents which induced granulocytic differentiation, such as 160 mM dimethyl sulfoxide and 100 nM retinoic acid, significantly decreased insulin receptor expression compared to monocytic inducing agents. This difference in insulin receptor expression correlated with binding characteristics in normal human peripheral granulocyte and monocytes. The HL-60 cell line offers a model for the study of the molecular events which lead to the contrasting insulin receptor expression during myeloid and monocytoid hematopoiesis.

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.

Different methods for determination of T-lymphocytes in human peripheral blood were compared: rosetting with sheep erythrocytes (SRBC), AET-treated SRBC, immunofluorescence using a monoclonal antibody against T cells (BL-T2), complement dependent cytolysis with polyclonal antisera against thymocytes, cytochemical demonstration of unspecific acid alpha-naphthyl-acetate esterase (ANAE), and electrophoretic mobility using a cell electrophoresis system (PARMOQUANT 2). Depending on the method, mean values between 70 and 79% T cells among separated mononuclear cells (MNC) were found. All paired observations were subjected to statistical analysis using rank correlation and U-test. From this analysis it is concluded that rosetting with SRBC, immunofluorescence using the monoclonal T-cell antibody and cytochemical reactivity for ANAE are favored methods for determining the T cell content of human MNC. However, the monoclonal antibody BL-T2 and the ANAE are not generally applicable because both markers were also found on malignant B-lymphocytes (B-CLL).

A supravital staining procedure for the identification of macrophages in cell suspension using a modification of a standard cytochemical assay for alpha-naphthyl acetate esterase (ANAE) activity is described. Macrophages are stained an intense red-brown after 5 min incubation in a buffer using ANAE as the substrate and hexazonium pararosaniline as the coupler for the azo dye. There is close agreement in the number of ANAE-positive cells found and the number of macrophages identified in smears by morphological criteria, by phagocytosis, and by the presence of Fc receptors. Therefore, this stain provides a quick, inexpensive method to estimate the number of macrophages present in suspensions of lymphocytic tissues from rats and mice.