Biomedical subjects
Publications and source records attributed to A Polliack.
A rare case of solitary CNS chloroma in a patient with chronic granulocytic leukemia (CGL) is described. This appears to be the first reported case of intracranial chloroma in CGL without systemic blastic crisis.
Megakaryoblasts and maturing megakaryocytic precursor cells from 5 patients with megakaryoblastic leukaemia were studied by scanning electron microscopy (SEM). The diagnosis in all cases had been established by ultrastructural cytochemistry on the basis of a positive platelet peroxidase reaction with negative staining for myeloperoxidase. 1 case presented as acute myelofibrosis and 4 as acute megakaryoblastic transformation of chronic granulocytic leukaemia. Under the SEM, megakaryoblasts and maturing megakaryocytic precursors showed typical surface features including the presence of rounded and irregular blebs, broad folds and pseudopodia. The nature of these surface blebs is still unclear but they probably represent surface membrane alterations relating to imminent platelet shedding at least in the more mature precursors. These surface microprojections are distinctly different from those encountered on leukaemic lymphoblasts, myeloblasts and monoblasts. It is suggested that SEM may be used in conjunction with the PPO reaction as in aid in the diagnosis of megakaryocytic leukaemias.
Selected lymphocytes from a patient with rheumatoid arthritis were infected with Epstein-Barr virus (EBV) and the emerging cell line (RF-AN) has now been successfully maintained in culture for more than two years. Multiparameter studies, including surface markers, ultra-structure and scanning immuno-electron microscopy, were utilized to characterize and evaluate this unique human cell line. Like other B-lymphoblastoid cell lines, RF-AN cells display multiple microvilli, contain surface and intracytoplasmic immunoglobulin (IgM lambda) and continuously produce monoclonal IgM lambda in vitro. The latter serves as an autoimmune antibody directed against IgG and is termed rheumatoid factor (RF). When RF-AN cells were incubated with human or rabbit IgG conjugated to polystyrene (latex) microspheres, almost all cells were labeled with the marker. However, cells did not label with goat IgG conjugated to latex. RF-AN cells were also labeled with latex microspheres coupled to goat antisera directed against human IgM or lambda chains but not with goat anti-human IgG or anti-human kappa chains. It is suggested that this mode of immunomicroscopy is reliable and may provide more useful data than other standard immunological techniques.
Prothymocytes were obtained from regenerating thymuses of intrathymic-irradiated, bone marrow-shielded rats. In contrast to cortical thymocytes, which are small nondividing cells containing nuclear TdT, prothymocytes are characterized by their large size, high mitotic activity, lack of natural attachment, absence of PNA-binding capacity, nonexpression of membranal thymic specific antigens, and absence of nuclear TdT. In addition, these cells are capable of responding to the mitogens Con-A and PHA, and are sensitive to in vitro lysis by physiologic concentrations of corticosterone and cortisol. Prothymocytes incubated for 3 days on thymic monolayers differentiated into small lymphocytes expressing cortical thymocyte characteristics. Light and electron microscopy studies demonstrated the infiltration of prothymocytes from the circulation via the thymic blood vessel wall into the perivascular sinuses. Prothymocytes isolated from the thymuses, however, did not exhibit specific "homing" to the thymus when transfused back into the animals. In view of the observed accelerated thymic repopulation in adrenalectomized rats, and the high in vitro glucocorticoid sensitivity of the prothymocytes, it is suggested that thymic homeostasis is regulated by specific effect of adrenocortical hormones on the prothymocyte subset.
In this report we review the use of scanning immuno-electron microscopy (SIEM) as a means of elucidating the location of cell surface membrane immunoglobulins (SMIg) and antigens (SMAg) on normal and leukemic human leukocytes. SMIg as well as SMAg were detected on various subpopulations of cells under the SEM, readily enabling identification of different types of circulating and cultured leukocytes. It is apparent from current data that the application of highly-specific monoclonal antibodies, multiple labeling procedures and improved methods of quantitation with SEM may well provide a more sensitive and elegant methodology for the identification of individual membrane components in a wide variety of mammalian cell systems.
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A patient with coexistent Gaucher disease and Philadelphia positive chronic granulocytic leukemia (CGL), who subsequently developed myeloblastic leukemia, is described. The diagnosis of CGL was established according to standard clinical, morphological, biochemical, and cytogenetic data, while the diagnosis of true Gaucher disease was based on biochemical data and the presence of Gaucher cells with typical ultrastructural features in the bone marrow and spleen. Enzyme studies showed low activity of ceramide-beta-glucosidase in the patient's peripheral blood leukocytes, skin fibroblasts, and splenic tissue and the presence of increased amounts of ceramide-beta-glucoside in the spleen. This case is reported in order to draw attention to the possible coexistence of these two diseases in the same patient, as opposed to the well-recognized finding of "Gaucher-like" cells in the bone marrow of patients with CGL. Enzyme studies enable distinction between these two situations.
Leukemic cells from the peripheral blood of 52 patients with acute and chronic leukemias were incubated with 12-0-tetradecanoyl phorbol ester (TPA). Thirty-one cases of lymphocytic leukemia (18 cases of acute lymphoblastic and 13 cases of chronic lymphocytic leukemia), 13 cases of acute nonlymphoblastic (myelo or myelomonoblastic) leukemia, and eight cases of blastic crisis of CGL (seven cases of predominantly myeloblastic crisis, and one case of lymphoblastic crisis) were studied. In all cases of lymphoid leukemia, cells formed clumps or aggregates after exposure to TPA, while in all cases of myeloid leukemia cells became adherent to the substrate. Seven of the eight cases of blastic crisis of CGL were predominantly myeloid in type and cells adhered to the substrate, while in a single case of lymphoid crisis in CGL cells formed clumps after TPA exposure. Functional, cytochemical, and ultrastructural studies showed altered cell differentiation and continuing in vitro maturation of leukemic cells after exposure to TPA. In the light of the above results, it is concluded that this simple test employing TPA exposure in vitro serves as a reliable means of distinguishing blasts from different origins in human leukemias.
The GDM-1 permanent cell line was established from the peripheral blood of a patient with a Philadelphia chromosome negative myeloproliferative disorder, after transformation to acute myelomonoblastic leukemia. The GDM-1 cells exhibited the same characteristics as those isolated from the peripheral blood of the patient prior to death: cells contained non-specific esterase sensitive to fluoride, myeloperoxidase, lysozyme (muramidase), and exhibited both Fc and complement (C3) receptors but lacked B- and T-cell surface markers including T-associated antigens. E-rosetting capacity, surface and intracytoplasmic immunoglobulins and EBV determined nuclear antigen (EBNA). The GDM-1 cells bore the 1a receptor and the myeloid leukemia antigen (M-1). The karyotype of the cultured leukemic cells showed the same specific chromosomal abnormalities present in the monoblasts obtained from the peripheral blood prior to death, indicating that the cell line was derived from the original leukemic cells.
A rare case of simultaneous presentation of monocytic and plasma cell leukemia is reported. Cytochemistry, transmission and scanning electron microscopy confirmed the presence of a dual population consisting of monoblasts and plasma cells. Monoblasts contained nonspecific esterases, secreted lysozyme and showed dense bodies and surface ruffles under the scanning electron microscope, while the plasma cells secreted IgG kappa paraprotein, contained rough endoplasmic reticulum, and showed surface blebs with microvilli. Another unusual feature of this case was the relatively chronic course of the disease, lasting 15 months after initial diagnosis.
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Human chronic lymphocytic leukemia (CLL) cells like prothymocytes and immunoactivated T-lymphocytes are readily lysed in vitro by pharmacological concentrations of glucocorticoids such as cortisol, whereas peripheral blood lymphocytes and thymocytes are unaffected by the hormone. In this study, metabolic and ultrastructural aspects of the cortisol-induced killing process of CLL cells are recorded. In vitro lysis was found to be temperature dependent and was detected only after 6 to 8 hr incubation with cortisol by means of the trypan blue exclusion test. However, 30 min of incubation with cortisol at either 37 degrees or 4 degrees followed by the removal of the hormone was still sufficient to induce the lytic process. Ultrastructural studies demonstrated sequential changes in the cytoplasm, including swelling of mitochondria and cytoplasmic decompartmentalization, followed by loss of surface microvilli with the appearance of "holes" in the cell membrane, and subsequent condensation of nuclear chromatin. The large holes in the membrane appearing after 6 hr of incubation with the hormone may be the cause for the penetration of the viable stain into the dead cells, as seen by light microscopy. Addition of metabolic inhibitors including actinomycin D, puromycin, and cycloheximide following administration of cortisol resulted in inhibition of the cell lysis. An excess of an antagonist such as cortexolone was found to inhibit the cortisol-induced cytolysis of the CLL cells. It is suggested that the glucocorticoid-induced lysis of human CLL cells is similar to the phenomenon observed in rat or murine lymphocytes and is mediated by interaction of the steroid molecule with the cytoplasmic receptor. The resulting complex appears to activate specific gene(s) the products of which eventually cause cytolysis.
Lymphocytes from the peripheral blood of patients with chronic lymphocytic leukemia (CLL) were examined for the presence of several recognized surface receptors [immunoglobulins, C3 component of complement, and concanavalin A (Con A)] and for structural changes in membrane distribution and dynamics, such as mobility and capping. There appeared to be a general decrease in the mobility of the various receptors on the surface membranes of CLL cells as compared with normal lymphocytes. Although C3 receptors were present on the majority of CLL cells, only a small proportion of the cells formed EAC rosettes. The response to phytohemagglutinin (PHA), Con A and pokeweed mitogen (PWM) confirmed previous observations that CLL cells respond poorly to the B cell mitogen, PWM, and that this response is significantly lower than that to the T cell mitogens, PHA and Con A. The decrease in response to all three mitogens seemed to correlate with the clinical stage of the disease, and it appears that Stage II cases may constitute a heterogeneous group of patients in this respect. A differential response to reduced concentrations of PHA was observed in CLL lymphocytes, but not with normal cells. The data concerning B and T cell function in CLL cells, as determined by the transformation and rosetting capacity of these cells, suggest a general decrease in function, which apparently deteriorates with progression of the disease.
Increased numbers of bone marrow mast cells were found in 45 (2.2%) of 2,000 bone marrow specimens obtained from patients who had hematologic disorders. Mast cells were most frequently seen in the marrows of patients who had preleukemic syndromes, lymphoproliferative disorders, and acute leukemia. The 16 patients who had preleukemic syndromes included those with refractory sideroblastic and megaloblastic anemia (with or without an excess of blasts), idiopathic pancytopenia or pure erythrocytic aplasia, paroxysmal nocturnal hemoglobinuria, idiopathic refractory neutropenia, agranulocytosis or thrombocytopenia, and persistent eosinophilia. Five of the seven patients who had acute leukemia had nonlymphoblastic leukemia; two had blastic crisis of chronic granulocytic leukemia. Of the 13 patients who had lymphoproliferative disorders, eight had chronic lymphocytic leukemia, three had macroglobulinemia, and two had non-Hodgkin's lymphoma. Three patients who had chronic renal failure associated with severe anemia and two who had chronic liver disease, splenomegaly, or hypersplenism were also encountered. In this study there appeared to be a consistent relationship between the presence of increased numbers of mast cells and the lymphocyte and plasma cell counts in the bone marrow. The significance of the presence of secondary mastocytosis in premalignant lesions, neoplasia, and, in particular, lympho- and myeloproliferative disorders, is still unclear.
Circulating plasma cells from six patients who had plasma cell leukemia were examined by transmission and scanning electron microscopy. In all cases, leukemic plasma cells constituted more than 60% of the total cell population in the peripheral blood. Transmission electron microscopy confirmed that the leukemic cells were plasmacytic and that many of them contained parallel arrays of rough endoplasmic reticulum and a prominent Golgi apparatus. Scanning electron microscopy confirmed previous observations of cultured myeloma cells and showed that plasma cells display varying numbers of surface blebs in addition to short stublike microvilli. The microvilli were frequently clustered together in one area of the surface. Bleb formation appears to be characteristic of plasma cells, but its nature is still obscure. Current knowledge of this phenomenon is briefly reviewed.
This report reviews the contribution of scanning electron microscopy (SEM) in haematology. Important observations regarding red cell shape and deformities are referred to and contributions in the definition and classification erythrocyte disorders are stressed. In this field there is no doubt that SEM has contributed much to the three-dimensional visualization of RBC disorders. As far as leucocyte pathology is concerned the situation is less clear. SEM has contributed much to current knowledge and understanding of the surface properties of white blood cells. Normal leucocytes have different surface features and can be distinguished under the SEM. However, some overlap does occur, making individual distinction on the basis of surface architecture alone extremely difficult. The difficulties in this regard are discussed in this review and factors influencing the variability of surface microprojections are reviewed briefly. Leukaemic cells of different origins may also be distinguished under the SEM. However, while "hairy' cells have typical surface features and non-lymphoid leukaemic cells, in particular monocytes, are readily distinguished from lymphoid leukaemic cells, there is much overlap of surface topography. Undifferentiated cells and early myeloblasts and lymphoblasts have similar surface features and cannot be distinguished under the SEM. While SEM adds a valuable third dimension to morphology and ultrastructure, it cannot be used alone in the definition of difficult cases of acute leukaemia.
In this study scanning immuno-electron microscopic (SIEM) techniques were used to identify human leukaemia-lymphoma cells. Monodispersed polystyrene (latex) beads were conjugated to specific antisera using glutaraldehyde, in an attempt to detect surface antigenic components on a variety of cells of known origin. Antisera, mostly immunoglobulin fractions, against human thymus (T) derived cells, common type acute lymphoblastic leukaemia cells (C/ALL) and surface immunoglobulin (sIg) bearing cells were used to coat latex spheres, while rabbit anti-mouse Thy-1 antiserum or whole human-IgG (gamma-globulin) bound to latex were used as controls in some experiments. The use of SIEM techniques in the direct mode as a simple and sensitive method for labelling surface antigens is described. The disadvantages of the SIEM methodology are also summarized while the requirements for optimal cell preparation using this technique are stressed. The experiments were designed to ascertain whether prolonged fixation of cells could be used prior to incubation of the cells with the marker. In this respect, repeated neutralization of the glutaraldehyde with glycine is essential. SIEM labelling of cells is random and unreliable without adequate quenching with glycine. The heteroantisera used in this study proved to be adequate and insignificant non-specific attachment and cross reactivity were seen. SIEM adds a further dimension to ultrastructural aspects of immunology and is a potentially useful tool in the study and identification of leukaemia and lymphoma cells.
Scanning immuno-electron microscopic labelling techniques were successfully used to identify a variety of leukaemia-lymphoma cells. Anti-T, -B and -common type acute lymphoblastic leukaemia (ALL) antisera were covalently conjugated to polystyrene latex spheres by means of glutaraldehyde. Peripheral blood mononuclear cells obtained from healthy individuals and from patients with T-type ALL, B-type chronic lymphocytic leukaemia (CLL), acute and chronic non-lymphoblastic leukaemias (ANLL) and cultured leukaemia-lymphoma cells were incubated with the above antisera-latex conjugates. Positively labelled cells were easily identified by the presence of latex spheres on their surfaces. Cross reactivity of the antisera used and non-specific attachment of the markers were infrequently encountered. The results of this study confirm earlier scanning electron microscopic reports indicating that different leukaemic cells have characteristic surface features and show that scanning immuno-electron microscopy (SIEM) can readily be applied to the study of human leukaemic cells.