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Immunohistochemical demonstration of leucocyte elastase in human tissues.

A polyclonal antiserum, produced in sheep and reactive against purified human leucocyte elastase, has been applied to paraffin sections from a range of human tissues by means of the indirect immunoperoxidase method. Striking and consistent results have been obtained. Normal or inflammatory granulocytes were intensely positive, the reaction being "blocked" by pure elastase. Activity was not seen in other sites, including histiocytic reticulum and lymphoid cells, although some weaker reaction was present in gastric and ileal lining epithelium. Strong reactivity was also seen in the cells of acute and chronic myeloid leukaemia and in extramedullary haemopoiesis. The advantages of this technique are compared with those for muramidase (lysozyme), alpha 1-antitrypsin, and naphthol-AS-D-chloroacetate esterase.

Granulocytes↗

Histochemical demonstration of chymotrypsin like serine esterases in mucosal mast cells in four species including man.

Serine esterases were detected in the granules of mucosal mast cells from rat, mouse, sheep, and man. Successful demonstration of enzyme activity required brief fixation (6 h) of tissues in 4% paraformaldehyde. Staining with naphthol AS-D chloroacetate produced an intense red reaction product in intraepithelial mucosal mast cells (globule leucocytes) and mucosal mast cells within the lamina propria of the gastrointestinal tract. The mast cell identity of cells stained for esterase was confirmed by sequential staining with toluidine blue (pH 0.5). Furthermore, the numbers of cells detected after staining for esterases or with toluidine blue were highly correlated. Esterase activity within mucosal mast cells/globule leucocytes from all species was inhibited with the serine enzyme inhibitor phenylmethylsulphonyl fluoride. Further histochemical studies with the substrate, N-acetyl-DL-phenylalanine B-naphthyl ester, indicated that mucosal mast cells and globule leucocytes contain esterases which are chymotrypsin like in substrate specificity.

Animals↗

Granulocytic sarcoma: misleading immunohistological staining with MT1 and S100 protein antibodies.

Tissue from two patients with granulocytic sarcomas stained positively for MT1 and S100 protein antibodies; both of these cases presented considerable clinical and histological diagnostic difficulties until acute myeloblastic leukaemia spread to the bone marrow. Tissues from a further eight patients with granulocytic sarcoma were also examined retrospectively. Seven of them stained for MT1 and four for S100 protein but the traditional histological markers for myeloid cells--chloroacetate esterase and lysosyme--often stained only weakly and focally. This pattern of staining should raise the possibility of a granulocytic sarcoma in otherwise problematic cases.

Adult↗

Granulocytic sarcoma of the cervix: an immunohistochemical, histochemical, and ultrastructural study.

Light microscopical and routine immunohistochemical studies of a cervical neoplasm in a 32 year old woman initially suggested a histiocytic lymphoma, but histochemical staining for chloroacetate esterase established the correct diagnosis. This was supported by electron microscopic findings. Eight months later the patient developed a granulocytic sarcoma in her left breast and haemotological features of acute myeloid leukaemia. Accurate initial diagnosis of granulocytic sarcoma in a non-leukaemic patient may reduce the risk of subsequent acute myeloid leukaemia if appropriate chemotherapy is begun in time.

Adult↗

Granulocytic sarcoma with expression of CD30.

A case of a young man with a spinal epidural tumour, initially diagnosed as large cell anaplastic malignant lymphoma, is reported. The tumour consisted of poorly differentiated cells showing immunoreactivity with antibodies directed against CD30 and CD45. Ten months later the patient developed acute myeloid leukaemia. The histological slides of the epidural tumour were reviewed, including additional enzymochemical and immunochemical stains. As the tumour showed immunoreactivity for myeloperoxidase and chloroacetate esterase, it was reclassified as a granulocytic sarcoma.

Adult↗

Childhood non-Hodgkin lymphomas in the United Kingdom: findings from the UK Children's Cancer Study Group.

AIM: To review the presenting clinical features and the histology of cases of non-Hodgkin lymphoma (NHL) entered into the United Kingdom Children's Cancer Study Group NHL Trial. METHODS: Sections of biopsy specimens from all cases entered into the trial were stained with Giemsa and haematoxylin and eosin. All cases were stained immunohistochemically for CD45, CD3, CD45RO, CD20, and CD30. Sections were stained with either naphthol AS-D chloroacetate esterase or KP1 (CD68) to identify granulocytic tumours. In a minority of cases, additional immunohistochemical stains were performed when necessary to establish the diagnosis. The sections were reviewed by three pathologists. RESULTS: Of 308 cases analysed, 293 were categorised as NHL. There was only one case of low grade lymphoma in the series. Over 80% of the cases fell into the categories Burkitt lymphoma (42.2%), lymphoblastic lymphoma (27.2%) and anaplastic large cell lymphoma (15.1%). Cases of Burkitt lymphoma presented most often with abdominal tumours mainly of the ileocaecal region. Tumours of the oropharynx and nasopharynx were also common in this group. Of the 84 lymphoblastic lymphomas, 56 were of the T-cell phenotype, 12 of the B-cell phenotype and 16 of indeterminate lineage. Most of the T-lymphoblastic lymphomas showed mediastinal or pleural involvement. Infiltration of the skin and soft tissues was seen in 25% of lymphoblastic lymphoma of B or indeterminate phenotype. Forty six children were diagnosed as having anaplastic large cell lymphoma, the majority being of T or indeterminate lineage. Most patients presented with lymphadenopathy but involvement of the bones, soft tissues or skin was seen in seven patients and of the mediastinum and lungs in five. CONCLUSION: Childhood non-Hodgkin lymphomas are almost all high grade and frequently extranodal. They fall mainly into the categories Burkitt lymphoma, lymphoblastic lymphoma and anaplastic large cell lymphoma. The separation of these subcategories can be made on the basis of morphology and immunohistochemical features. The anatomical distribution of these different categories of non-Hodgkin lymphoma is distinctive.

Adolescent↗

Obstructive uropathy associated with myelomonocytic infiltration of the prostate.

A 72 year old man was diagnosed with chronic myelomonocytic leukaemia (CMML) according to the FAB group classification. He presented with symptoms of anaemia, urinary frequency, hesitancy, and nocturia. He was later admitted with acute urinary retention and acute renal failure, which resolved with treatment. A transurethral resection of the prostate was performed. Histological examination showed fibromuscular hyperplasia with dense infiltration by myelomonocytes which stained positively with chloroacetate esterase; immunohistochemical staining was positive for lysozyme, CD43, CD45, and CD68. Following treatment with oral etoposide he transformed to acute myeloid leukaemia and eventually died. Myelomonocytic infiltration of the prostate has not been reported before. This case extends the spectrum of disease previously recognised in CMML.

Aged↗

Granulocytic sarcoma of the rectum: a rare complication of myelodysplasia.

A 67 year old man with myelodysplasia was admitted as an emergency with a six week history of rectal bleeding and diarrhoea. Barium enema showed an irregular polypoid filling defect in the lateral wall of the proximal rectum near the rectosigmoid junction. Histology showed this to be a granulocytic sarcoma (extramedullary granulocytic leukaemia; chloroma) infiltrating the bowel. A low index of suspicion of this lesion results in an incorrect diagnosis in many such cases. A chloroacetate esterase immunoperoxidase stain will confirm the granulocytic nature of the tumour cells.

Aged↗

Focal lymphoid aggregates (nodules) in bone marrow biopsies: differentiation between benign hyperplasia and malignant lymphoma--a practical guideline.

AIMS: To provide practical guidelines for the differentiation between benign and malignant focal lymphoid aggregates (lymphoid nodules) in routinely referred bone marrow trephine biopsies, using a synoptic approach including clinical data and histological workup. METHODS: For easy identification of very small lymphoid infiltrates the chloroacetate esterase stain was applied as a screening procedure. This allowed the identification of 491 formalin fixed, paraffin wax embedded specimens with one or more lymphoid nodules. Examination of lymphoid infiltrates included such variables as histotopography, demarcation, cytology, reticulin fibres, and immunohistochemistry with a set of monoclonal antibodies (CD20, CD45R, CD45R0, CD3, CD43). Evaluation of clinical and morphological data was carried out independently. In case of malignant lymphomas, a correlation with corresponding lymph node findings was made. RESULTS: 352 patients had benign focal lymphoid aggregates usually associated with systemic autoimmune diseases, chronic myeloproliferative disorders, toxic myelopathy, and viral infections. Discrete nodular infiltrates of (small cell) malignant lymphomas (n = 93) simulating benign hyperplasia were found in chronic lymphocytic leukaemia, germinal centre cell lymphomas (CB-CC), and lymphoplasmacytic/cytoid lymphomas (LPI). In addition to immunoreactivity, certain histological variables proved distinctive. These were: (1) histotopography, that is, localisation of the lymphoid aggregates within the bone marrow space; (2) relation to the surrounding tissue: margination or interstitial spillage of lymphoid cells; and (3) increase in reticulin fibres. CONCLUSIONS: A combined diagnostic procedure identifying several distinctive features, in particular histotopography and immunohistochemistry, provides a most promising way of discriminating reactive from neoplastic lymphoid nodules in the bone marrow.

Bone Marrow↗

Detection of c-kit mutation Asp 816 to Val in microdissected bone marrow infiltrates in a case of systemic mastocytosis associated with chronic myelomonocytic leukaemia.

BACKGROUND/AIMS: The occurrence of myeloid leukaemia in patients with systemic mastocytosis is a well recognised phenomenon. However, the pathophysiological basis of such a coevolution has not been clarified. Recent data have shown that the c-kit mutation Asp 816 to Val is detectable in neoplastic mast cells in most patients with systemic mastocytosis, including those who have associated haematological disorders. The aim of this study was to study clonal disease evolution by analysing bone marrow cells from a patient with systemic mastocytosis and associated chronic myelomonocytic leukaemia (CMML) for the presence of this mutation. METHODS: The DNA of microdissected bone marrow cells from a patient with systemic mastocytosis and associated CMML was analysed for the presence of the c-kit mutation Asp 816 to Val by means of HinfI digestion and direct sequencing of semi-nested polymerase chain reaction (PCR) products. RESULTS: The two neoplasms could easily be identified and discriminated in paraffin wax embedded bone marrow sections by tryptase and chloroacetate esterase staining. A total number of 10 tryptase positive systemic mastocytosis infiltrates and 10 tryptase negative CMML infiltrates were removed by microdissection. As assessed by HinfI digestion and direct sequencing of semi-nested PCR products, the c-kit mutation Asp 816 to Val was detected in five of seven systemic mastocytosis infiltrates and four of six CMML infiltrates. By contrast, no c-kit mutation Asp 816 to Val was found in bone marrow infiltrates in patients with CMML without associated systemic mastocytosis (n = 20). CONCLUSION: These data support a monoclonal evolution of systemic mastocytosis and concurrent CMML in the patient studied.

Aged↗

Indoxyl-beta-D-glucuronide, a novel chromogenic reagent for the specific detection and enumeration of Escherichia coli in environmental samples.

About 97% of Escherichia coli strains produce beta-glucuronidase, but almost all other Enterobacteriaceae lack this enzyme. A D-glucopyranosiduronic acid (glucuronide) possessing a readily detectable beta-linked aglycone should, therefore, constitute a specific reagent for the detection of this organism. For this purpose, the title compound has been synthesized for the first time. The synthesis proceeds in eight steps from readily available D-glucuronolactone, anthranilic acid, and chloroacetic acid and can be carried out on a large scale. The compound has the predicted properties: when included in the standard membrane filter test for the analysis of water, indoxyl-beta-D-glucuronide allows specific detection of E. coli through the formation of blue colonies that are the result of rapid conversion of the liberated aglycone to indigo. The recovery of E. coli is easily measured and almost quantitative.

Chemical Phenomena↗

Neonatal mastocytosis with pachydermic bullous skin without c-Kit 816 mutation.

BACKGROUND: Bullous mastocytosis is an unusual variant of mast cell disease with widespread bullae as the main cutaneous feature induced by mast cell proteases that cause dermoepidermal separation. CASE REPORT: A rare case of diffuse cutaneous bullous mastocytosis with pachydermia and unusually extensive skin folding is described in a 3-week-old girl. The diagnosis was confirmed by immunohistochemistry with Giemsa stain, the naphthol ASD chloroacetate esterase reaction and elevated blood levels for tryptase, histamine in serum and histamine and 1.4-methylimidazol acetic acid in the 24-hour urine. Blood cell count was normal, as were thrombocytosis and leukocytosis. FACS analysis of the bone marrow aspiration material showed 1% mast cells. No c-Kit 816 [Asp-->Val] somatic mutation was found. Systemic involvement of other organs was excluded. DISCUSSION: The prognosis of c-Kit-negative diffuse bullous mastocytosis is not known. Regular blood controls are mandatory, and screening for germ cell ovarian cancer and bone marrow controls should be performed as well.

Female↗

Cytochemical and immunophenotypic heterogeneity in acute promyelocytic leukemia.

The leukemic promyelocytes in 37 cases of acute promyelocytic leukemia (APML; FAB, M3) were examined for their cytochemical property. Thirty-two cases (86%) showed strong myeloperoxidase (MPO), chloroacetate esterase (Es-chl) and Sudan black B (SBB) positivity, suggesting a pure neutrophilic differentiation of the leukemic cells. However, in 5 out of 37 cases, a strong, diffuse alpha-naphthyl acetate esterase (Es-a) positivity, which was sensitive to sodium fluoride treatment was observed in addition to strong MPO, Es-chl and SBB positivity. This suggested monocytic differentiation of a proportion of APML cases. In 31 cases, surface marker studies were carried out with the help of a panel of monoclonal antibodies consisting of two panmyeloid antibodies (GM 58/8 and 1G10), one anti-HLA-DR antibody (7.2) and one 'myeloid' antibody (5F1) with restricted reactivity with monocytes (CD14). The purpose of including the monoclonal antibodies 5F1 and 7.2 was to determine if a correlation could be established between strong Es-a positivity and reactivity of the leukemic promyelocytes with 5F1 and 7.2 in individual cases. All the 5 cases with 'monocytoid' cytochemistry were unreactive with 5F1, and only one case in this group showed 15% 7.2-positive cells. The lack of immunophenotypic support for the monocytic cytochemistry of the 5 cases of APML suggests that the monocytic phenotype of leukemic promyelocytes is both aberrant and incomplete. Since normal promyelocytes are purely neutrophilic, this could be a manifestation of an 'intralineage infidelity' in APML, similar to that observed in other types of acute leukemia. The clinical significance of 'monocytic' phenotype of APML is not clear; cases with monocytic differentiation did not show different clinical and hematological features when compared to the more common, pure neutrophilic variety.

Antibodies, Monoclonal↗

Multiple granulocytic sarcoma during complete hematologic remission of acute nonlymphoid leukemia.

In this report we describe a case of granulocytic sarcoma (GS) developing in a patient after 66 months in complete remission of acute nonlymphoid leukemia. The granulocytic precursor cell proliferation arose simultaneously in two extramedullary sites (testis and small bowel), without evidence of bone marrow relapse. The intensive systemic chemotherapy with high-dose ARA-C allowed a clinical remission, lasting 8 months. GS eventually recurred as extramedullary multi-site disease (abdominal lymph nodes, central nervous system involvement), once again unassociated with blood and bone marrow relapse. Both, at onset and in relapse, special staining techniques on tissue sections (chloroacetate esterase) and immunohistochemical typing with monoclonal antibodies were necessary for a correct diagnosis. We discuss the relationship between systemic leukemia and the GS which showed a metastasizing tumor-like behavior.

Humans↗

Defective terminal maturation along monocyte-macrophage lineage in chronic myelomonocytic leukemia.

The maturation process of leukemic myelomonocytoid cells derived from peripheral blood of two typical cases of chronic myelomonocytic leukemia was investigated in vitro. In both cases, liquid culture of the leukemic cells in the presence or absence of 12-o-tetradecanoyl phorbol-13-acetate yielded either monocyte-macrophages or mature granulocytes, respectively, when examined by May-Giemsa staining. However, using cytochemical criteria, both of these mature cell fractions were found to be heterogenous and still contained substantial numbers of cells positive for both nonspecific and chloroacetate esterases. Phagocytic activity was markedly defective in the monocyte-macrophages recovered from culture as well as in freshly-isolated peripheral monocytes in both patients. In addition, active oxygen production from isolated monocytes stimulated by zymosan was also decreased in one of the cases. The results indicate that at least in some cases of chronic myelomonocytic leukemia, leukemic cells have the potential to differentiate in vitro to monocyte-macrophages which, however, show only limited development of functions, as well as freshly-isolated monocytes from peripheral blood.

Aged↗

Intraepithelial migration of nasal mucosal mast cells in hay fever.

Mast cells were studied by light microscopy in mucosal imprints and in biopsies of nasal mucosa of 12 birch pollen allergic individuals before and during the pollen season, using techniques optimized for the demonstration of mucosal mast cells. We also measured the histamine content of nasal mucosa, whole blood and plasma, and counted the numbers of circulating blood basophils. Before the pollen season the nasal mucosa was found to contain many mast cells located in the mucosal connective tissue stroma, and very few cells with basophilic and metachromatic granules were found in mucosal imprints. During the pollen season there was a redistribution of mast cells into the epithelium, many such cells now being recovered in mucosal imprints. The total number of mucosal mast cells counted in tissue sections did not change significantly with the onset of the pollen season, suggesting a redistribution of mucosal mast cells by migration. Judged by morphologic appearance and naphthol-AS-D chloroacetate esterase activity, the intraepithelial mast cells found in tissue sections had rather the properties of tissue mast cells than of blood basophils, and only a few of the basophilic cells of the imprints had a morphology compatible with blood basophils. The histamine content of the mucosa, as well as histamine levels of whole blood and plasma, and circulating blood basophil numbers did not change significantly in relation to the pollen season. These findings suggest that an intraepithelial migration of mucosal mast cells is part of the allergic mucosal response.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

Characterization of basophil-like cells derived from nonhuman primate bone marrow.

In order to determine whether mast cells or basophils could be derived from nonhuman primate bone marrow, cells from bone marrow aspirates were cultured in the presence of concanavalin A-stimulated nonhuman primate spleen cell supernatants (CAS). Culture conditions were identical to those used for culturing mucosal-like mast cells from mouse bone marrow. In this situation, basophil-like cells (BLC) could be identified in liquid cultures and averaged 14-19 microM in size, were round or oval in appearance, had lobulated nuclei, and contained less than 100 metachromatically staining granules per cell. By electron microscopy, granules had dense oval or semilunar cores with surrounding fibrous whorls. BLC were peroxidase positive, chloroacetate esterase negative, stained positively with acid toluidine blue, and contained 0.1-0.3 pg histamine per cell. BLC expressed IgE receptors and were Leu 5b and Leu 16 negative. IgE-sensitized BLC released histamine after stimulation with antihuman IgE or the calcium ionophore A23187. [35S]-labeled proteoglycans were degraded with chondroitinase ABC but not with heparinase, indicating the absence of heparin in BLC. Thus, culture conditions that include the use of CAS and lead to the growth of mast cells from rodent bone marrow result in the growth of BLC from nonhuman primate bone marrow. These observations suggest that fundamental differences exist in the type of histamine containing cells that arise from rodent and primate bone marrow when such bone marrow cells are cultured under identical conditions.

Animals↗