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E de Harven

Publications and source records attributed to E de Harven.

At least 19 recordsLinked to original sources
PubMed1996

Functional and morphological characterization of immunomagnetically selected CD34+ hematopoietic progenitor cells.

We evaluated the potential of immunomagnetically selected (miniMACS) progenitor cells to give rise to colony-forming cells and their precursors, detected as long-term culture-initiating cells (LTC-IC), as well as their capacity to expand in liquid cultures. A 90% mean purity, a 43.2% yield and a 55.8-fold enrichment were achieved from normal bone marrow. When corrected for enrichment, the mean number of committed progenitor cells and the frequency of LTC-IC (evaluated by means of limiting dilution assay [LDA]) were not statistically different in low density mononuclear cells or in the CD34-enriched fractions. In five cases CD34+ selected cells grown in a stroma-free long-term bone marrow culture system with the addition of stem cell factor, interleukin 3, interleukin 6 and GM-CSF every 48 h, showed a 15 (+/- 15) and 31 (+/- 21) mean colony forming unit-granulocyte/macrophage fold increase on cultures at days 7 and 14. However, when corrected for enrichment, the expansion capability of these cells was significantly lower than that of the unseparated fraction, particularly after the first week. Immediately after separation, electron microscopy revealed that the CD34+ selected fraction contained more than 45% of well-differentiated myeloid cells (MPO+), with iron beads preferentially clustered at one pole of the cell surface and sometimes already endocytosed in pinocytic vesicles. After 24 h and 48 h incubation at 37 degrees C, the majority of the cells showed no iron particles, but about 30% of the cells were iron-labeled phagocytic cells. The percentage of apoptotic cells with internalized iron was negligible. These data show that immunomagnetically separated CD34+ cells may have a slightly impaired short-term expansion capability, but give rise to both committed and more primitive progenitor cells. During the separation, the iron beads are internalized, rapidly processed in the cytoplasm and do not seem to interfere with in vitro growth.

Adult↗
PubMed1995

Immunogold labeling for the diagnosis of leukemia by transmission and scanning electron microscopy.

For the cell type diagnosis of leukemia in adult patients, particularly when the sampling of bone marrow is difficult, the study of peripheral blood leukocytes (PBLs) by immuno-electron microscopy provides significant information, as illustrated here in two cases of hairy cell leukemia and seven cases tentatively identified as megakaryoblastic leukemia (M7). Indirect immunogold labeling with the B-ly7 monoclonal antibody (CD103) proved valuable in confirming the diagnosis of hairy cell leukemia. Immunogold labeling for the GpIIIa platelet glycoprotein (CD61) was used in cases where the light microscopy of blood films revealed possible megakaryoblastic leukemia. Under the electron microscope, however, the CD61 positive cells showed, in almost all cases, a wide spectrum of megakaryopoietic differentiation which made the diagnosis of M7 questionable. Most of the CD61 positive cells featured cytoplasmic differentiation markers such as alpha granules and demarcation membranes, further confirming the presence of circulating megakaryocythemia, a phenomenon described many years ago in various myeloproliferative disorders. It is suggested, therefore, that many of these cases should not be identified as true megakaryoblastic leukemias.

Humans↗
PubMed1994

Immunogold labelling of leukemic hairy cells with the B-ly7 monoclonal antibody: an SEM and TEM study.

Two cases of hairy cell leukemia have been studied by immuno-TEM and immuno-SEM after immunogold labelling of the cell surface antigen recognized by the B-ly7 monoclonal antibody. Most hairy cells appeared significantly labeled, although the density of the expression of the antigen, as demonstrated by immunogold labelling, seems variable from cell to cell. Moreover, some cells with the morphology of hairy cells and which could not be identified as monocytes were not labeled. Labelling for the antigen identified by the B-ly7 mAb does not seem to correlate with the presence of ribosome lamellae complexes which were present only in one of the two cases studied. Rare lymphocytes of unidentified lineage were labeled. Monocytes were significantly absent from the samples of peripheral blood of the two patients studied. In one normal control sample, monocytes were observed unlabelled. The results are discussed in reference to the pathogenesis of hairy cell leukemia, its surprisingly low mitotic rate, and its distinct response to chemotherapy.

Adult↗
PubMed1994

Cell type identification in leukemia: the level of agreement among four independent diagnostic methods.

The level of agreement among four independent diagnostic methods for cell-type identification in leukemia was examined. The four diagnostic methods were routine morphology, electron microscopy, cell surface marker analysis by flow cytometry, and cancer cytogenetics. Sixty-five blood and bone marrow samples from fifty-seven patients were independently investigated by the four methods. It was found that when all cell-type categories were taken into consideration, overall percentages of agreement for pairs of the four diagnostic methods were poor, ranging from 43% to 67%. The kappa statistics, which correct for the agreement expected by chance, were also calculated. These kappa statistics had a range from 0.17 to 0.40 when all cell-type categories were taken into consideration, again indicating poor agreement between the methods pairwise. When each cell type was considered separately, much of the observed agreement was found to be expected on the basis of chance, as indicated by many of the kappa statistics being 0.00. For all categories, overall agreement for all four methods was poor and statistically significant, after correcting for chance (kappa = 0.20, (p = 0.000046). The poor agreement found among the four diagnostic methods indicates the need for further validation studies.

Cytodiagnosis↗
PubMed1992

Antibody drug carrier for immunotherapy of superficial bladder cancer: ultrastructural studies.

Superficial bladder cancer represents a promising target for intravesical, antibody-guided therapy. The construction of an optimum antibody-cytotoxic drug conjugate depends mostly on the appropriate selection of a monoclonal antibody (mAb). We have used immunogold labeling and SEM to specifically map the distribution of antigens expressed on three bladder cancer cell lines and on the luminal surface of biopsies from human transitional cell carcinoma of various grades and from normal bladder mucosa. The 48-127 mAb, which recognizes a M(r) 54,000 surface glycoprotein (gp54), was found to be very promising as a potential drug carrier. This antibody reacts with the surface of cells from low- and high-grade tumors; it does not react with the normal urothelium. Labeling of normal bladder mucosa was observed, however, on microvillous intermediate urothelial cells occasionally exposed by small areas of desquamation. The 48-127 mAb could target drugs to all areas of transformed urothelium while avoiding drug delivery to the normal, undesquamated bladder mucosa. Kinetics of gp54/48-127/gold complexes were tested in vitro with T24 and RT4 human bladder carcinoma cell lines incubated in the presence of the 48-127 mAb directly conjugated with 17.7-nm gold particles. Internalization of the gp54/48-127/gold complex was readily demonstrated by transmission electron microscopy. These results suggest that the 48-127 mAb represents a valuable drug carrier for intravesical therapy, allowing specific tumor targeting and internalization of various cytotoxic agents.

Aged↗
PubMed1990

Immunogold labeling of the low-affinity (55 kd) IL2 receptor on the surface of IL2 receptor-bearing cultured cells and mitogen-activated peripheral blood lymphocytes.

Foreign antigens or mitogens initiate activation of T lymphocytes through antigen-specific receptors. After antigen-receptor interaction, T cells release a lymphotrophic hormone, interleukin 2 (IL2), and high-affinity IL2 receptors (IL2R) are progressively expressed on their surfaces. When evaluated under the scanning electron microscope after specific immunolabeling with colloidal gold markers, the density of expression of the 55 kd low-affinity IL2 receptor (Tac) increases in a time-dependent manner, reaching a maximum 72 hours after the onset of phytohemagglutinin (PHA) activation. In contrast, the number of cells expressing the 55 kd molecule reaches a maximum by 24 hours and remains constant through 72 hours. Immunogold labeling allows simultaneous determination of IL2 receptor density at the level of each individual cell and of the percentage of IL2 receptor-positive cells in any cell population under study. The rank order of receptor expression on the surface of established culture cell lines correlates with results reported from other laboratories with immunofluorescence and Scatchard analysis of binding of radiolabeled IL2. Although at high density of receptor expression immunogold labeling with 40 nm gold markers detects significantly fewer sites than radiolabeling, the method appears very sensitive in identifying low levels of expression. This results from 1) the extremely low non-specific background of the immunogold labeling technique and 2) the analysis of single cells rather than cell populations. Our results indicate that 2 cell lines, reported as negative for the expression of the 55 kd IL2 receptor (YT2C2 and MLA144), express low numbers of this molecule as demonstrated by immunogold labeling.

Antibodies, Monoclonal↗
PubMed1990

Scanning electron microscopy of immuno-gold labeled antigens associated with bladder cancer.

Scanning electron microscopy (SEM), in the backscattered electron imaging (BEI) mode, has been used to study the topographical distribution of colloidal gold labeled antigens expressed on the luminal surface of the bladder urothelium in biopsies from three categories of patients: 1) normal controls; 2) patients with a history of bladder cancer but no pathological diagnosis at time of cystoscopy; and 3) patients with overt transitional cell carcinoma (TCC) of various histopathological stages and grades. Cold cup biopsies were processed for immuno-SEM according to a previously described method. Antigens under investigation were: 1) ABH blood group antigens; and 2) those identified by the following murine monoclonal antibodies (mAbs): LEU-M1, T16, 19A211, G4 and E7. In most cases labeling patterns were correlated with the surface features of the superficial urothelial cells as revealed in the secondary electron imaging (SEI) mode of the SEM. Results, to date, indicate that the immuno-gold labeling method is more sensitive than immuno-peroxidase, and that phenotypic heterogeneity of antigenic expression (or deletion) is a frequent observation of potential diagnostic or prognostic value.

ABO Blood-Group System↗
PubMed1987

Scanning electron microscopy in the backscattered electron imaging (BEI) mode: applications to clinical hematology.

Elements of high atomic number backscatter electrons that carry information of cytochemical and of immunocytochemical significance in high resolution scanning electron microscopy (SEM). Reaction products of enzyme cytochemistry containing lead or osmium have been used to localize the sites of phosphatase and of endogenous peroxidase, respectively. Particles of colloidal gold, ranging in diameter from 40 to 13 nm, have been successfully used to label cell surface antigens specifically identified by murine monoclonal antibodies. The diagnostic potential of the scanning electron microscope in clinical hematology appears to be considerably enhanced by the use of the backscattered electron imaging (BEI) mode. Quantitation of the number of gold-labeled epitopes on cell surfaces cannot be achieved in the conventional secondary electron mode of the SEM, but is an attractive possibility in the backscattered electron imaging mode.

Antigens, Surface↗
PubMed1987

Immuno-scanning electron microscopy of normal and leukemic leukocytes labeled with colloidal gold.

The immunogold method, utilizing 40 nm colloidal gold particles which can be selectively visualized with the scanning electron microscope (SEM) in the backscattered electron imaging mode was used for the study of blood cells incubated with various monoclonal antibodies. Numerous anti-leukocyte monoclonal antibodies still recognize lightly glutaraldehyde prefixed antigens and can be used to identify various blood cell types and even to recognize their different maturation stages. Clearcut differences in surface morphology exist among peripheral blood normal leukocytes and even among the principal lymphocyte subclasses. Marked heterogeneity in surface morphology is, on the other hand, evident when studying precursors or leukemic cells. Immature cells show, nevertheless, relatively smooth surfaces while some distinct surface features appear on cells already committed toward a specific differentiation lineage. Hairy cells can also be precisely identified, especially when in small number in heterogeneous populations, combining their typical surface morphology with their positivity for B1 and Leu M5 monoclonal antibodies.

B-Lymphocytes↗
PubMed1987

Immunogold labeling of human leukocytes for scanning electron microscopy and light microscopy: quantitative aspects of the methodology.

When cell surface antigens are labeled with the colloidal gold marker, back-scattered electron images (BEI) reveal all the gold particles and, therefore, permit total counts. Secondary electron images (SEI) show only a small percentage of the gold particles and are inadequate for quantitative evaluation. For determination of the cellular labeling index, a time-consuming method implies the screening of 100 cells by scanning electron microscopy, at a magnification of approximately 12,000 to 15,000x, with continuous SE/BE shifts. A much more efficient method is to transfer the SEM sample or its equivalent under the light microscope and to count the total number of gold labeled cells in the epi-polarization mode. The total cell count can be evaluated under UV light, taking advantage of the autofluorescence of the glutaraldehyde fixed cells.

Antigens, Surface↗
PubMed1985

Scanning electron microscope cytochemistry of blood cells.

The backscattered electron imaging (BEI) mode of scanning electron microscopy (SEM) has been applied to study various histo-cytochemical reactions in biological specimens since the early seventies. Due to numerous, recent technical improvements the BEI mode of SEM now belongs to the routine of many SEM laboratories. For cytochemistry, BEI has been mainly used to: visualize intracellular structures and organelles; recognize the different cell types in heterogeneous populations or tissues; study the correlations between enzymatic activities and cell surface features. We have evaluated the most relevant results obtained in the study of blood cells and the possible future applications of these techniques.

Blood Cells↗
PubMed1984

A novel approach for scanning electron microscopy of colloidal gold-labeled cell surfaces.

A method is described for the use of scanning electron microscopy on the surface of gold-labeled cells. It includes the use of 45- or 20-nm colloidal gold marker conjugated with Staphylococcal protein A. The marker is best recognized on the basis of its atomic number contrast by using the backscattered electron imaging mode of the scanning electron microscope. When the backscattered electron signal is mixed with the secondary electron signal, an optimum correlation between the distribution of the labeled sites and the cell surface structures is demonstrated. The method is illustrated by its application to the identification of human circulating granulocytes. Its good resolution, high contrast, and good labeling efficiency offers a promising approach to the specific localization of cell surface antigenic sites labeled with particles of colloidal gold.

Erythrocytes↗
PubMed1984

Scanning electron microscopy of human leukocytes.

Extensive application of scanning electron microscopy (SEM) to the study of normal as well as of pathological human leukocytes has been pursued over the last ten years. Initial efforts to recognize, under the SEM, the two main subpopulations of circulating lymphocytes have met with limited success. However, a critical evaluation of the cell preparatory methods has emerged from the initial difficulties. Highly reproducible SEM images of leukocytes can now be obtained, and their significance correlated with immunological and/or cytochemical parameters. This resulted from the development of highly specific techniques for the immuno-labeling of cell surfaces, and from the development of SEM cytochemistry in the backscattered electron imaging (BEI) mode. A basic procedure for the SEI imaging of separated peripheral blood leukocytes (PBL's) is critically presented with the hope of facilitating significant future applications of SEM in diagnostic clinical hematology.

Cell Membrane↗
PubMed1983

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.

Acid Phosphatase↗