Chloroacetate Esterase Staining Research Articles

Multimarker immunohistochemical staining of calgranulins, chloroacetate esterase, and S100 for simultaneous demonstration of inflammatory cells on paraffin sections.

Mac387 monoclonal antibody (MAb) recognizes two calcium binding, myeloid-associated proteins, now termed calgranulins, expressed at high levels by neutrophils and monocytes. Calgranulins are related to migration inhibitory factor (MIF) and are lost in a few days from monocytes differentiated in vitro. This marker is therefore potentially useful to analyze macrophage heterogeneity and turnover in tissue sections. In this study, we developed an immunohistochemical multimarker technique, including calgranulin demonstration, suitable for analyzing different inflammatory cells on paraffin-embedded material. The technique was carried out in subsequent steps demonstrating (a) naphthol AS-D chloroacetate esterase (CAE); (b) S100 immunoreactivity using a rabbit antibody in peroxidase-antiperoxidase (PAP) staining; and (c) Mac387 immunoreactivity using the alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique. CAE staining was introduced in this method to distinguish Mac387+/CAE- macrophages from Mac387+/CAE+ neutrophils, and Mac387-/CAE+ mast cells. S100 protein is strongly expressed within lymphoid tissues by dendritic accessory cells and was then applied to distinguish these cells from S100-macrophages. We have also verified the possibility of reducing the staining time for this time-consuming procedure by use of microwave irradiation. The technique was applied to a representative variety of normal and pathological samples to assess its usefulness for study of cell heterogeneity. Our results showed the multimarker technique to be highly informative in the study of inflammatory lesions (e.g., rheumatoid arthritis, sarcoid and cat-scratch granulomas, dermathopathic lymphadenopathy), and is of wide potential value as an aid to histopathological diagnosis of several diseases.

Biphenotypic Acute Leukemia in Adults

Leukemias are characterized by an idiopathic proliferation of a progenitor cell that is committed to a single cell lineage. However, leukemias with dual-lineage differentiation are being described, especially within the pediatric age group. The authors reviewed 118 cases of adult acute leukemia phenotyped by immunofluorescent flow cytometry; 7 cases demonstrated mixed cell lineage. Immunophenotypically these cases were defined by early B-lymphocyte differentiation (TdT, HLA-DR, and CD19) coexpressed with a myeloid receptor (CD13, CD15, or CD33) on the same leukemic cell. Routine cytochemical evaluation demonstrated punctate positivity of the blasts with naphthol AS-D chloroacetate esterase stain in five of seven cases. Cytogenetic analysis revealed structural abnormalities of chromosome 11 in four of the seven cases. Three of these studies showed a break at 11q23-24, the location of the human proto-oncogene ets-1. Clinically, two of these leukemias represented chronic myelogenous leukemia in blast crisis, and all cases behaved aggressively. The authors' data suggest that mixed lineage leukemias are an identifiable subset of adult acute leukemias and are associated with a poor prognosis.

BETA-glucuronidase and chloroacetate-esterase staining discriminates rat liver sinusoidal endothelial cells from Kupffer cells in primary culture

Beta-glucuronidase and N-AS-D-chloroacetate esterase cytochemistry have been applied to rat liver sinusoidal endothelial cells and Kupffer cells. Both staining procedures allowed a clear-cut differentiation of either cell type. Kupffer cells which had been stained with beta-glucuronidase showed a positive reaction, whereas sinusoidal endothelial cells were completely negative. If the chloroacetate reaction was used, the former stained diffusely while the latter showed a characteristic granular staining pattern. Identity and purity of sinusoidal endothelial cells and Kupffer cells was validated by transmission and scanning electron microscopy as well as by the pattern of released eicosanoids which is characteristic for either cell type. These two staining techniques are a valuable addition to the peroxidase reaction commonly applied for differentiation.

Use of all-trans retinoic acid in the treatment of acute promyelocytic leukemia

Twenty-four patients with acute promyelocytic leukemia (APL) were treated with all-trans retinoic acid (45 to 100 mg/m2/day). Of these, eight cases had been either nonresponsive or resistant to previous chemotherapy; the other 16 cases were previously untreated. All patients attained complete remission without developing bone marrow hypoplasia. Bone marrow suspension cultures were studied in 15 of the 24 patients. Fourteen of these patients had morphological maturation in response to the retinoic acid (1 mumol/L). Chloroacetate esterase and alpha-naphthyl acetate esterase staining as well as electronmicroscopic examination confirmed that retinoic acid-induced cells differentiated to granulocytes with increased functional maturation (as measured by nitroblue tetrazolium reduction, NBT). The single nonresponder to retinoic acid in vitro was resistant to treatment with retinoic acid but attained complete remission after addition of low-dose cytosine arabinoside (ara-C). During the course of therapy, none of the patients showed any abnormalities in the coagulation parameters we measured, suggesting an absence of any subclinical disseminated intravascular coagulation. The only side effects consisted of mild dryness of the lips and skin, with occasional headaches and digestive symptoms. Eight patients have relapsed after 2 to 5 months of complete remission. The others remain in complete remission at 1+ to 11+ months and are still being followed up. We conclude that all-trans retinoic acid is an effective inducer for attaining complete remission in APL.

Use of All-Trans Retinoic Acid in the Treatment of Acute Promyelocytic Leukemia

Twenty-four patients with acute promyelocytic leukemia (APL) were treated with all-trans retinoic acid (45 to 100 mg/m2/day). Of these, eight cases had been either nonresponsive or resistant to previous chemotherapy; the other 16 cases were previously untreated. All patients attained complete remission without developing bone marrow hypoplasia. Bone marrow suspension cultures were studied in 15 of the 24 patients. Fourteen of these patients had morphological maturation in response to the retinoic acid (1 mumol/L). Chloroacetate esterase and alpha-naphthyl acetate esterase staining as well as electronmicroscopic examination confirmed that retinoic acid-induced cells differentiated to granulocytes with increased functional maturation (as measured by nitroblue tetrazolium reduction, NBT). The single nonresponder to retinoic acid in vitro was resistant to treatment with retinoic acid but attained complete remission after addition of low-dose cytosine arabinoside (ara-C). During the course of therapy, none of the patients showed any abnormalities in the coagulation parameters we measured, suggesting an absence of any subclinical disseminated intravascular coagulation. The only side effects consisted of mild dryness of the lips and skin, with occasional headaches and digestive symptoms. Eight patients have relapsed after 2 to 5 months of complete remission. The others remain in complete remission at 1+ to 11+ months and are still being followed up. We conclude that all-trans retinoic acid is an effective inducer for attaining complete remission in APL.

Involvement of tumor necrosis factor in endotoxin-triggered neutrophil adherence to sinusoidal endothelial cells of mouse liver and its modulation in acute phase

Tumor necrosis factor (TNF) has been shown to mediate lipopolysaccharide-induced neutrophil adhesion to liver sinusoidal endothelium in vivo. Female NMRI mice received either 5 micrograms lipopolysaccharide (R595) per animal alone (model A) or together with 116 mumol D-galactosamine (model B). One hour after injection, TNF activity in the serum was detectable to an equal extent in both models. Neutrophils in the liver, which had been identified by chloroacetate esterase staining of liver sections and quantitated by light microscopy, started to increase at 1 h and were elevated 10-fold above baseline at 6 h after application in (A) and (B). If 0.5 micrograms TNF instead of lipopolysaccharide was injected alone (model C) or together with D-galactosamine (model D), neutrophil influx into the liver was comparable to that observed in (A) or (B). Alanine aminotransferase activity in the serum was nearly normal in (A) and (C) 6 h after injection, while it reached levels up to 50-fold above baseline in models (B) and (D). This reflects the well-known D-galactosamine sensitization against lipopolysaccharide or TNF. Furthermore, degranulation of a large number of intrasinusoidal neutrophils could be observed 9 h after lipopolysaccharide-galactosamine injection. The administration of 116 mumol D-galactosamine per animal alone led neither to a measurable TNF activity in the serum nor to an increase in alanine aminotransferase activity or number of liver neutrophils. If the animals had received 50 microliter turpentine subcutaneously 24 h prior to lipopolysaccharide, TNF or D-galactosamine injection, the induced acute-phase reaction suppressed the increase of liver neutrophils in all models. Acute-phase reaction also prevented neutrophil degranulation and the rise of alanine aminotransferase in (B) to a great extent, while serum TNF activity was only minimally affected. It is concluded that TNF mediates neutrophil adhesion to the sinusoidal endothelium in vivo and that acute-phase reactants prevent lipopolysaccharide- or TNF-induced neutrophil influx into the liver.

Clinico-pathological study on denture stomatitis.

The clinical and pathological study was performed in order to determine the histopathological and cytoimmunological characteristics of denture stomatitis. All specimens were biopsy materials from seventeen patients with denture stomatitis. Normal palatal mucosae from ten patients served as the control. In addition to the usual staining methods, naphtol AS-D chloroacetate esterase stain and peroxidase-antiperoxidase method were used to detect mast cells and plasma cells. Denture stomatitis could be divided into atrophic and hyperplastic types. The former showed a smooth and atrophic mucosa. The latter showed a large number of exophytic projections which were composed of marked acanthosis and submucosal fibrosis, and was further subdivided into granular and papillary subtype according to the size of projections. In the present study, there were six cases of the atrophic type, and eleven cases of the hyperplastic type (consisting of seven granular and four papillary subtypes). The hyperplastic type was more frequently observed in patients with partial dentures compared with complete dentures and was associated frequently with ill fitting of the denture base as well as agglutination of denture plaque. Cytoimmunological study revealed that there was a pronounced increase of plasma cells, especially IgG- and IgA-producing cells, and a moderate increase of lymphocytes as well as mast cells in both types of denture stomatitis. Mast cells were always noted in the area with marked plasma cell infiltration, suggesting an intimate relation between both cells. These findings suggest that the immunological reactions play some role in the pathogenesis of denture stomatitis.

Release of granulocyte-specific colony-stimulating activity by human bone marrow exposed to phorbol esters

Granulocyte-macrophage colony growth depends on the presence of colony- stimulating activity (CSA). Phorbol esters induce concentration- dependent colony formation in the absence of exogenous CSA. We questioned whether phorbol esters mimicked the action of CSA by directly stimulating colony growth, or whether phorbol esters acted indirectly by inducing marrow cells to release CSA. First, after incubating human bone marrow cells with phorbol 12,13-dibutyrate (PDB) for 3 days, we separated PDB from the protein peak of the conditioned medium by Sephadex G-10 gel filtration and tested this peak for the presence of CSA. When diluted 1:10 in the agar colony assay, this material induced 133 +/- 15 colonies/10(5) bone marrow cells. Second, to determine whether bone marrow cells required the continued presence of PDB in order to release CSA, PDB was removed from bone marrow cells by washing, and these cells were reincubated in fresh medium in the absence of PDB. CSA was found in the medium of these cultures; its release was maximal after preincubation of bone marrow cells with 5 X 10(-8) M PDB for 3 days, followed by incubation for 3 days in the absence of PDB. This CSA stimulated granulopoiesis out of proportion to monocytopoiesis, with 85% +/- 17% of the colonies being granulocytic (as indicated by histochemical staining for chloroacetate esterase), and 12% +/- 3% being monocytic (as indicated by nonspecific esterase). Inhibitors of monocyte colony formation, including PGE1, were not present in the medium that contained this CSA. These studies demonstrate that normal human bone marrow cells exposed to PDB release CSA and that this CSA selectively stimulates granulopoiesis in vitro.

Release of Granulocyte-Specific Colony-Stimulating Activity by Human Bone Marrow Exposed to Phorbol Esters

AbstractGranulocyte-macrophage colony growth depends on the presence of colony-stimulating activity (CSA). Phorbol esters induce concentration-dependent colony formation in the absence of exogenous CSA. We questioned whether phorbol esters mimicked the action of CSA by directly stimulating colony growth, or whether phorbol esters acted indirectly by inducing marrow cells to release CSA. First, after incubating human bone marrow cells with phorbol 12,13-dibutyrate (PDP) for 3 days, we separated PDB from the protein peak of the conditioned medium by Sephadex G-10 gel filtration and tested this peak for the presence of CSA. When diluted 1:10 in the agar colony assay, this material induced 133 ± 15 colonies/105 bone marrow cells. Second, to determine whether bone marrow cells required the continued presence of PDB in order to release CSA, PDB was removed from bone marrow cells by washing, and these cells were reincubated in fresh medium in the absence of PDB. CSA was found in the medium of these cultures; its release was maximal after preincubation of bone marrow cells with 5 × 10−8M PDB for 3 days, followed by incubation for 3 days in the absence of PDB. This CSA stimulated granulopoiesis out of proportion to monocytopoiesis, with 85% ± 17% of the colonies being granulocytic (as indicated by histochemical staining for chloroacetate esterase), and 12% ± 3% being monocytic (as indicated by nonspecific esterase). Inhibitors of monocyte colony formation, including PGE1, were not present in the medium that contained this CSA. These studies demonstrate that normal human bone marrow cells exposed to PDB release CSA and that this CSA selectively stimulates granulopoiesis in vitro.

Cytochemical determination of granulocyte elastase and chymotrypsin in human myeloid cells and its application in acquired deficiency states and diagnosis of myeloid leukemia.

Two cytochemical methods for detection of granulocytic elastase and chymotrypsin employing alanine and phenylalanine naphthyl esters were developed. Specificity of reaction with the ester substrates was proven by chloromethyl ketone inhibitors. The results of both staining methods were almost identical with the staining for naphthol AS-D chloroacetate (Cl Ac-O Nap AS-D) esterase, since Cl Ac-O Nap AS-D also reacts with granulocyte elastase and chymotrypsin. Mature neutrophils and myeloid precursors except myeloblasts are stained with all three substrates in peripheral blood and bone marrow. Mast cells, however, only react with Cl Ac-O Nap AS-D and the chymotrypsin substrate and not with the elastase substrate. In acute myeloid leukemia the three esterases appear in parallel at a somewhat later stage of maturation than myeloperoxidase. In blood smears from 380 hospital patients no hereditary elastase or chymotrypsin deficiency could be demonstrated. Staining for elastase and chymotrypsin was also normal in hereditary myeloperoxidase deficiency and chronic granulomatous disease. On the other hand 6% of the hospital patients and about two-thirds of patients with acute myeloid leukemia showed a partial elastase deficiency in more than 25% of the peripheral neutrophils.

In vitro generation of mast cell-like cells from human peripheral mononuclear phagocytes.

An in vitro culture method, previously shown to induce the transformation of rat macrophages to mast cells, has been adapted to humans. Adherent peripheral human blood mononuclear cells were cultured in a medium supplemented with 30% L-cell supernatant and 30% horse serum. During the first week of culture, the cells were phagocytic (80%), lacked histamine, were not metachromatic and stained faintly for naphthol-AS-D-chloroacetate esterase, primarily in a paranuclear location. During the first 2 weeks of culture, the cells grew in size, synthesized DNA, became nonadherent and developed cytoplasmic chloroacetate esterase staining which was strongly positive in 50-60% of the cells during the third week. Intracellular histamine levels increased steadily during culture, and electron-dense metachromatic cytoplasmic granules were present during the third week. Human peripheral blood mononuclear cells contain therefore a subpopulation of cells that can differentiate into mast cells-like cells under specific in vitro culture conditions.

Nonlymphoid hematopoietic malignant disease of the lymph nodes

The extension of myeloproliferative malignant tumors to lymph nodes was studied in seven excision biopsies and 27 autopsies in which lymph nodes were obtained from all nodal regions. We observed the proliferation of poorly, partially, and well differentiated neoplastic cells with a predominance of immature cells of the granulocytic series and fewer cells of the erythroid and megakaryocytic cell lines. Disturbances of the lymph node architecture consisted of invasion by abnormal cells of the trabecular stroma, distrupting the reticulin mesh and extending toward the pericapsular area. In the lymph node a loose network of thin and thick fibers replaced the orginal framework. Generalized lymph node involvement at autopsy showed preservation of the architecture, both in cases with total replacement of the lymphoid population by abnormal cells and in cases with involvement by single cells or small groups of cells. Partial involvement occurred chiefly in the medullary zone, probably the result of hematogenous dissemination from the vascular plexus in medullary cords. Further abnormal cell development was linked to trabecular infiltration. The extensive involvement seen at autopsy took place by lymphatic dissemination. In lymph nodes studied at autopsy an abnormal immunoblastic reaction was observed. At first these abnormal cells were suspected of representing the myeloproliferative malignant disease, but the application of special staining techniques revealed their polyclonal cytoplasmic immunoglobulins. The chloroacetate esterase stain and the immunohistochemical stains for muramidase and hemoglobulin were especially useful in demonstrating that myeloproliferative diseases develop in the environment found in lymph nodes.

The thymus as haematopoietic tissue of non-lymphoid cells.

Non-lymphoid haematopoiesis in the thymus was studied in 33 cases ranging from 14 weeks gestation up to 15 months postnatally. All thymuses displayed focally granulopoiesis in the connective tissue septa and in the lymphoid tissue, where it was localized in the peripheral cortex and at the cortico-medullary junction. Within the lymphoid tissue the granulocytic series showed many precursor cells with large rounded nuclei. Their origin could only be identified by the naphtol AS D chloroacetate esterase stain. In cases with thymic atrophy these precursor cells were present in relatively large numbers. Within the connective tissue septa many granulocytes with nuclear segmentation were intermingled with precursor cells. The erythroid series were detected with an immunohistochemical method for demonstration of haemoglobin. The positively stained nucleated cells occurred mainly in nests mostly localized in the peripheral cortex. These precursor cells were usually found in different places from the granulopoietic cells. No megakaryocytes were found. With appropriate techniques non-lymphoid haematopoiesis can be demonstrated in the developing thymus and must be considered as an expression of normal growth.

Clinical-pathological findings and cytochemical characterization of myelomonocytic leukaemia in 5 dogs

The initial clinical signs of 5 dogs diagnosed to have myelomonocytic leukaemia lacked specificity and one dog was asymptomatic. Clinical findings were variable but splenomegaly or spleno-hepatomegaly was detected. All dogs had either macrocytic or normocytic normochromic, non-responsive anaemia and thrombocytopenia. The total leukocyte count ranged from 10 400 to 422 400 per μl of blood with 4 dogs having increased numbers. Cellular morphology in Wright-stained blood and marrow films revealed the presence of predominantly myeloblasts with or without monoblastoid and monocytoid cells and indicated a diagnosis of myeloblastic leukaemia in 2 cases and of myelomonocytic (blast type) in 3 cases. However, cytochemical characterization of cells in blood and marrow smears for granulocytic (alkaline phosphatase) and monocytic (lipase and non-specific esterase) markers led to all being classified as myelomonocytic (blast type) leukaemia. The number of cells reacting positively for these markers was invariably increased in blood, in marrow, or in both. In this regard, staining for peroxidase, chloroacetate esterase, phospholipid, and glycogen was found to have no or limited differential value. The clinical outcome was poor. Because of the advanced stage of the disease, 2 dogs were not treated and 2 of the 3 dogs given anti-cancer treatment survived for less than 2 weeks. One dog responded objectively to cytoreductive therapy and survived 8 months. Myelomonocytic transformation of an essentially monoblastic leukaemia is thought to have occurred in this case over a period of 3 months while on maintenance therapy with fatal outcome 5 months later.