Plasma membranes purified from myeloid leukemia cells before and after differentiation. I. Characterization of spectrin-like proteins and increased association of actin.

Two-step sucrose density gradient centrifugation was used to isolate the plasma membrane of a myeloid leukemia cell line (Ml). Calspectin (or fodrin) was identified in the Triton-insoluble fraction from the plasma membrane, and the molecular size and actin- and calmodulin-binding activity were studied. During differentiation of this cell line, which accompanied the induction of cell motility and phagocytic activity, the membrane-bound actin increased dramatically, whereas calspectin increased only slightly. Therefore, calspectin does not appear to have a major function in the increased binding of actin filaments to the plasma membrane, a requirement for the induction of cell motility.

Ultrastructural evaluation of periodate-reactive glycoconjugates in human leukaemia cells.

Periodate-reactive glycoconjugates in human leukaemic cells were examined electron microscopically by the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method. Granules in ALL cells were classified into 4 types based on PA-TCH-SP staining features. Abnormal granules containing glycogen were observed only in children with treatment-resistant ALL. Cytoplasmic granules in leukaemic cells of patients with AML and acute monocytic leukaemia exhibited moderate reactivity. The distribution pattern of glycogen in the cytoplasm of leukaemic cells was classified into 3 types, one lacking glycogen, one containing small glycogen particles scattered throughout cytoplasm, and one showing clusters of glycogen particles. Cells with glycogen clusters were observed in ALL cells and in erythroblasts from patients with erythroleukaemia. PA-TCH-SP reactivity was detected in the rough endoplasmic reticulum in acute promyelocytic leukaemia but not in ALL or other types of AML. Megakaryoblasts in megakaryocytic crisis of chronic myelogenous leukaemia exhibited characteristic PA-TCH-SP reactivity similar to that of normal megakaryocytes.

Megakaryoblastic transformation of Ph positive chronic granulocytic leukemia.

A case of well-documented and illustrated megakaryoblastic transformation in a patient with chronic granulocytic leukemia is presented. The salient features of this case were the presence of megakaryoblasts in the peripheral blood and bone marrow and characteristic cytochemical and electron microscopic findings. In addition, the authors observed an unusual, previously unreported, similarity of the abnormal platelets with those described in the Gray platelet syndrome. A literature review of the 13 previously described cases is included.

Immunologic, biochemical, and ultrastructural characterization of the leukemia cell in F344 rats.

Immunologic, biochemical, and morphologic characteristics of the mononuclear cell from the leukemia of F344 rats were determined. The cells were morphologically similar to large granular lymphocytes (LGL). Surface marker analysis revealed Fc gamma receptors, no Fc gamma receptor or complement receptor activity, and an inability to spontaneously rosette guinea pig erythrocytes. Leukemia cells also had a surface immunoglobulin that hemagglutinated normal rat erythrocytes. The surface immunoglobulin and Fc gamma receptors dissociated from the cell after 2 hours of in vitro incubation, but Fc gamma receptor activity was reexpressed after 6 hours of in vitro incubation. Cells were capable of adherence to glass surfaces but had a low capacity for phagocytosis of latex beads. Cytochemical analysis revealed a consistent, strongly positive reaction for esterase that was sensitive to NaF. The cytochemical profile of the leukemia cell was similar to that described for LGL.

Eosinophilic myelocytoma in an owl monkey (Aotus trivirgatus).

A mass found in the anterior mediastinum at necropsy of an adult female owl monkey (Aotus trivirgatus) was morphologically consistent with an eosinophilic myelocytoma. Lymph nodes, kidneys, bone marrow, and other tissues were diffusely infiltrated by neoplastic cells. Intracellular and extracellular Charcot-Leyden crystals were present in the neoplasm.

Scanning electron microscope cytochemistry of normal and leukaemic leukocytes.

Backscattered Electron Imaging (BEI) is a particular technique which permits to study cytochemical reactions with the Scanning Electron Microscope (SEM). The BEI data pertaining to specific enzymatic activities can be directly correlated to the surface morphology of each individual cell. Leukocytes from 5 normal individuals, 14 patients with acute nonlymphoblastic leukaemia (ANLL), 7 patients with chronic myeloid leukaemia (CML) and 3 patients with acute lymphoblastic leukaemia (ALL) were studied for myeloperoxidase activity, acid phosphatase localization, silver staining of the nuclei and phagocytosis of iron carbonyl in the BEI mode of SEM. Some normal peripheral blood leukocytes which cannot be distinguished by their surface morphology alone were satisfactorily identified with the BEI technique. Leukaemic myeloid cells can be recognized in many cases because of their positive myeloperoxidase reaction, while monocytic elements can be characterized by the presence of surface ruffles, acid phosphatase activity and active phagocytosis. The usefulness of the BEI technique in identifying different blood cell types with the SEM and its possible application to the diagnosis of certain cases of leukaemia are discussed.

Granulocytic sarcoma of the peritoneum.

Granulocytic sarcoma is an unusual extramedullary tumor composed of immature cells of the myelogenous series; it usually occurs during the course of myelogenous leukemia. This paper presents a rare case of granulocytic sarcoma of the peritoneum, occurring without evidence of myelogenous leukemia in peripheral blood and bone marrow.

Cytogenetic analysis of leukaemic colonies from acute and chronic myelogenous leukaemia.

We have utilized the blast cell assay of Buick et al. (1977) to grow and subsequently cytogenetically analyze cultured colony forming cells (CFUs) from patients with acute and chronic myelogenous leukaemia (AML, CML). Cytogenetic analysis of CFUs was successful in 30/36 cases (83%), a success rate similar to direct harvesting techniques. Identical clonal chromosomal abnormalities demonstrated by direct techniques were also observed in CFUs from AML and CML. Removal of T-precursor cells by E-rosetting prior to plating did not eliminate growth of karyotypically normal cells. The combination of morphologic and cytogenetic studies performed clearly established that the assay system supports the growth of leukaemic progenitors. Although both karyotypically normal and abnormal leukaemic colonies grew in this assay, growth of leukaemic colonies was much more likely if the plated cells were karyotypically abnormal (P = 0.010). Leukaemic colony growth was also more frequent if the tritiated thymidine labelling index (LI%) of plated cells was greater than or equal to 5% (P = 0.018). Leukaemic colonies were most likely (P = 0.018) to have been derived from plated cells with both abnormal karyotype and high LI% (greater than or equal to 5%). Cytogenetic analyses from cultured cells revealed only those karyotypic features found in the uncultured cells (i.e., no additional abnormal sublines were found). However, in most cases, the greatly enhanced number and quality of mitotic figures allowed for more detailed banding analysis.

Granulocytic sarcoma of the brain after renal transplantation.

A case of granulocytic sarcoma of the brain in a renal transplant recipient treated with immunosuppressive agents is presented. While the increased risk of malignant lymphoma, particularly large-cell lymphoma (reticulum cell sarcoma, histiocytic lymphoma, immunoblastic sarcoma) in these patients is well known, this appears to be the first report of a granulocytic sarcoma. Granulocytic sarcoma, a rare tumor composed of immature granulocytic elements, almost never involves the parenchyma of the brain. Histochemical examination may be necessary to distinguish this lesion from other poorly-differentiated neoplasms.

Variations in the activity of nucleolar organizers in different tissues, demonstrated by silver staining of human normal and leukemic cells.

A simple silver-staining technique that demonstrates those nucleolar organizing regions of metaphase chromosomes which are transcriptionally active during the preceding interphase (AgNORs) has been applied to cells obtained from the bone marrow and mitogen-stimulated peripheral blood lymphocyte cultures of hematologically normal individuals and patients with various forms of leukemia. In the majority of bone marrow cells from the normal controls and many of the patients, the number of cells with detectable AgNORs, and the staining intensities in those cells which were Ag+, were markedly reduced compared with the levels found in blood lymphocytes. The numbers of cells having satellite associations and the numbers of chromosomes participating in these associations also generally reflected the proportions of AgNORs present. When patterns of bone marrow silver staining were compared between patients with leukemia, distinct differences were found which could be correlated with cytology. It is suggested that different cell types have characteristic AgNOR staining profiles, reflecting specific regulation of ribosomal RNA synthesis in particular cell lineages. AgNOR staining may indicate, therefore, the predominant cell types that divide in the bone marrows of patients with different forms of leukemia.

3. The value of chromosome studies in the classification and management of the leukemias.

Chromosome identification techniques have shown the non-random nature of cytogenetic changes in leukemia. In addition, they have identified structural chromosome abnormalities occurring in specific types of acute leukemia as classified by the FAB criteria. Such cytogenetic sub groups are associated with differing prognoses. In acute lymphocytic leukemia, even the ploidy of the leukemic cells appears important in predicting prognosis. Identification of the Philadelphia (Ph1) chromosome has diagnostic significance in chronic granulocytic leukemia. This makes possible the classification of patients into Ph1 + and Ph1 - varieties which have different responses to therapy. Similar variations are found between the 2 groups of patients who do or do not develop additional abnormalities with blastic transformation. The implication of these findings in both acute and chronic leukemia may influence choice of therapy in the future.

Granulocytic sarcoma (chloroma) initially seen as acute mastoiditis.

Granulocytic sarcoma (chloroma) is a localized tumefaction of immature granulocytes that is typically seen in association with myelogenous leukemia. The primitive cell population seen in biopsy material may be misinterpreted as histiocytic lymphoma or other sarcoma unless additional studies are performed. We saw a 36-year-old woman with promyelocytic leukemia in remission who had the signs and symptoms of an acute coalescent mastoiditis. Histologic examination of the surgical specimen, however, demonstrated a granulocytic sarcoma. Our case exemplifies some of the difficulties that may be encountered in the diagnosis of granulocytic sarcoma and illustrates the point that symptoms of an inflammatory process in a patient with a diagnosis of leukemia must be regarded with a degree of suspicion.

Megakaryocyte cultures in the chronic phase and in the blast crisis of chronic myeloid leukaemia: studies on the differentiation of the megakaryocyte progenitors and on the maturation of megakaryocytes in vitro.

Megakaryocyte (MK) colony formation has been studied in the chronic phase and in the blast crisis of chronic myeloid leukaemia (CML). Blood cells were grown in plasma clot for 13 d. MKs were subsequently identified by immunofluorescent techniques using two monoclonal antiplatelet antibodies (AN51 and J15). The maturation process was studied by ultrastructural methods. A marked increase in the number of circulating CFU-MK was observed in all the 10 cases studied prior to chemotherapy (70-fold increase per ml of blood). No significant modification in the regulation of MK colony formation as compared to that of normal subjects was observed. The predominant abnormality in maturation in culture was the occurrence of many hypoploid MKs (microMKs). However, the cytoplasmic maturation of the MKs was identical to that of normal subjects with occasional platelet shedding. Since microMKs predominated in some patients, scoring of MK colonies in CML necessitated immunofluorescent labelling to permit identification of MKs. During the blast crisis, MK colony formation occurred in four out of five patients with an extremely high plating efficiency in the case of promegakaryoblastic transformation. In contrast, MK colonies could not be grown from blood samples of patients with acute leukaemia, including two cases of promegakaryoblastic leukaemia. Maturation of MKs in blast crisis was identical to that of the chronic phase. Furthermore, after short periods of culture in liquid medium, circulating promegakaryoblasts from patients in blast crisis matured with the consequent production of alpha-granules and demarcation membranes. These results confirm the contention that CML represents a pluripotent stem-cell disease, involving the MK lineage, and suggest that the block in maturation during the acute phase can be overcome in vitro.

Functional characterization of the cells in chronic neutrophilic leukemia.

Light and electron microscopy of neutrophils from chronic neutrophilic leukemia (CNL) did not reveal differences from normal mature neutrophils. However, functional characterization of CNL cells showed marked differences when compared to normal cells. CNL neutrophils were much less viable in suboptimal conditions. Their survival was further reduced by autologous serum and was corrected by normal human serm. CNL cells showed very active phagocytosis, but their bactericidal activity was reduced in suboptimal conditions. The total content of lysozyme and beta-glucuronidase was lower in CNL cells compared to normal neutrophils, but the release of these enzymes from stimulated cells was much higher than normal. This observation is compatible with a marked lysosomal lability. Cells from the patients' peripheral blood and bone marrow showed excessive growth in CFU-C assays. Marked susceptibility of CNL cells to cytotoxic activity of cold agglutinins, SLE sera, and CSFs was observed and may signify qualitative and/or quantitative differences in the membrane structure of CNL neutrophils, as compared to normal cells.