Ultrastructural features of CD34+ hematopoietic progenitor cells from bone marrow, peripheral blood and umbilical cord blood.

Hematopoietic progenitor cells from different sources have been widely characterized, but their ultrastructural morphology has never been described in detail. In this study, imunomagnetically separated CD34+ cells from normal bone marrow (BM), mobilized peripheral blood (PBSC) and human umbilical cord blood (CB) were studied by transmission electron microscopy (TEM) using a cytochemical method which reveals endogenous myelo-peroxidase (MPO) activity. This technique is particularly suited for detecting early signs of the myeloid commitment. The CD34+ cells from PBSC were morphologically very homogeneous and 94.7+/-4.5% of these cells were MPO-: these ultrastructural features are generally considered typical of immature cells. The CD34+ BM cells were instead more heterogeneous, with 24.6+/-7.4% showing intense MPO activity. The ultrastructural characteristics of CB cells fell between those observed in PBSC and BM, but there was a high percentage of morphologically immature cells with no evidence of MPO activity (about 83%). The number of apoptotic cells within samples from different sources was also examined both by TEM and flow cytometry. The percentage of apoptotic cells was 0.7% in PBSC, 2.3% in BM, 2.9% in CB from vaginal delivery and 11.6% in CB from cesarean section. These observations confirm the relative phenotypic immaturity of CB in comparison with BM cells; they also suggest that CB collected after cesarean section may be associated with reduced stem cells viability.

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.

Ultrastructural studies of the megakaryoblastic leukemias of Down syndrome.

The ultrastructure of the leukemic cells in transient leukemia (six cases), myelodysplasia (five cases) and acute megakaryoblastic leukemia (one case) in patients with Down syndrome were studied. The cells were identified to be of megakaryocytic lineage by virtue of the expression of platelet glycoprotein GpIIIa, detected by immunogold labelling. In all patients, some of the leukemic cells had ultrastructural features of megakaryocytes, including ectoplasmic protrusions, demarcation membranes, and alpha granules. Differentiation was greatest in the cells of patients with transient leukemia. These studies provide a detailed assessment of the ultrastructural features of the leukemic cells in the megakaryoblastic leukemias of Down syndrome.

Cell surface changes of hemopoietic cells during normal and leukemic differentiation: an immuno-scanning electron microscopy study.

Hemopoietic cells display a wide range of cell surface antigens which are either lineage specific or acquired during differentiation. Monoclonal antibodies can be used, in conjunction with colloidal gold markers, to identify under the scanning electron microscopy (SEM) at the single cell level, specific lineage or maturation stages in the hemopoietic bone marrow. Normal bone marrow cells, either gradient separated or purified by immuno-magnetic methods and leukemic cell samples, which can be considered as "frozen" stages of hemopoietic differentiation, have been studied with this method. Typical cell surface morphologies, which characterize immature progenitor cells and cells committed or differentiated towards the lymphoid, myeloid, erythroid and megakaryocytic lineage have been identified. Correlations between cell surface features and some hemopoietic cells functions have been attempted on the basis of these findings.

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.

Immuno-electron microscopy characterization of human bone marrow stromal cells with anti-NGFR antibodies.

Human bone marrow stromal cells have been examined with an immuno-electron microscopy technique in order to better define their structure and function in normal hematopoiesis. Bone marrow fragments from normal donors, after mild permeabilization and glutaraldehyde prefixation were labeled with the Me20.4 Mab, which recognizes the low affinity nerve growth factor (NGFR) and was recently described as specifically identifying fibroblastic-like bone marrow stromal cells. Five nm gold immuno-conjugates served as markers. NGFR+ cells were showing either a star-shape, with long and convoluted dendritic projections, and branching with each other to form a complex system of lacunae upon which hematopoietic cells were arranged. Other NGFR+ cells had an elongated spindle-like morphology. NGFR+ dendrites were seen in close contact with each other and with the different hematopoietic cells, although definite junctions were never noticed. NGFR+ dendrites were also observed surrounding mature plasma cells, in close apposition with adipocytes or surrounding bone marrow sinusoids. These findings may give some clues about the function of the bone marrow stromal cells, which are known to be involved in the homing and recirculation of hemopoietic cells; in addition, the presence and distribution of NGFR in the bone marrow stroma may support the recent evidence of a co-stimulatory effect of NGF in early hematopoiesis.

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.

Myelodysplasia and acute megakaryoblastic leukemia in Down's syndrome.

In this report we describe the clinical and hematologic features of 23 cases of myelodysplasia (MDS) or acute megakaryoblastic leukemia (AMKL) occurring in Down's syndrome. MDS was characterized by thrombocytopenia, abnormal megakaryocytopoiesis, megakaryoblasts (< 30%) in the marrow and abnormal karyotype, the most common of which was trisomy 8, found in 7/15 patients with MDS. Three of five patients achieved a complete remission with low dose cytosine arabinoside, vincristine and retinyl palmitate. The high cure rate and the distinctive features of the leukemic process in these cases suggest that this type of MDS and AMKL are unique to patients with Down's syndrome.

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.

Human chromosomes: evaluation of processing techniques for scanning electron microscopy.

Methods for scanning electron microscopy (SEM) of chromosomes have been developed in the last two decades. Technical limitations in the study of human chromosomes, however, have hindered the routine use of SEM in clinical and experimental human cytogenetics. We compared different methodologies, including metal impregnation, air drying and specimen coating. SEM preparation of human chromosomes in which osmium impregnation is mediated by tannic acid, yielded more reproducible results when compared with osmium impregnation protocols previously described. The level of osmium impregnation was systematically evaluated by imaging chromosomes in the backscattering mode. Critical point drying and a light gold-palladium coating were essential for appropriate secondary electron imaging of chromosomes. With this method, and in a preliminary quantitative analysis, we show that our SEM technique is more sensitive than light microscopy for the detection of aphidicolin-induced fragile sites. This technical approach is useful for chromosomal studies requiring resolution higher than that obtained by light microscopy. Also, it allows the use of clinical and archival chromosomal samples prepared by routine cytogenetic techniques.

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.

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.

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.

Scanning electron microscopic study of immunogold-labeled human leukocytes.

For many years critical point drying (CPD) has been the method of choice for preparing cells for scanning electron microscopy (SEM). Described herein is a simple, efficient, inexpensive, reproducible, and safe procedure using Peldri II, a proprietary fluorocarbon compound that is solid at room temperature and a liquid above 25 degrees C, as a sublimation dehydrant for processing specimens for SEM. The utility of Peldri II was demonstrated in studies using leukocytes from the blood of healthy donors and patients with leukemia as well as from long-term lymphoblastoid cell lines. The application of the proposed Peldri II procedure was further documented in SEM studies in which the expression and distribution of the interleukin-2 receptor (IL-2R) on leukocyte surface membranes was imaged using colloidal gold-labeled antibodies (i.e., immunogold). When compared with current SEM preparation procedures using CPD, Peldri II is a useful alternative that is thought to offer several important advantages.

Double labelling of cell surface antigens with colloidal gold markers.

Particles of colloidal gold of different diameters (15 nm and 40) have been used to distinctively label different surface antigens expressed on the surface of human peripheral blood B and T lymphocytes. Silver enhancement has been used to facilitate the observation of the gold particles. Observations were carried out in the backscattered electron imaging mode of the scanning electron microscope. Two different methods have been compared: in Method I the two antigens have been identified by monoclonal antibodies of different classes (IgG and IgM); in Method II monoclonal antibodies of the same subclass were used but the ligands were different (goat anti-murine Ig versus biotin/streptavidin). Some cross-reactivity was observed with Method I, but not with Method II.

Malignant properties of sublines selected from a human bladder cancer cell line that contains an activated c-Ha-ras oncogene.

The human bladder cancer cell line MGH-U1 (also designated T-24 or EJ) contains an activated c-Ha-ras oncogene, which is amplified as compared to normal human fibroblasts. We have generated sublines from the MGH-U1 cell line: the MGH-U1/OCI subline was generated by dissociating spheroids formed from MGH-U1 cells; the U1-m/F1 and OCI-m/F1 were generated by in vivo passage of experimental lung metastases formed after i.v. injection of MGH-U1 and MGH-U1/OCI lines into immune-deprived mice; the U1/t subline was generated by in vivo passage of i.m. tumors formed from MGH-U1 cells. All sublines formed tumors in immune-deprived mice from smaller i.m. inocula than the parent line, and the U1-m/F1 subline generated more spontaneous metastases in lungs. Lung colony forming efficiency after i.v. injections of cells into similar mice was also greater for the sublines than for the parent MGH-U1 cells. The U1-m/F1 and OCI-m/F1 were the most tumorigenic lines. Early passages of the MGH-U1/OCI subline showed the presence of double minute chromosomes, and amplification and increased expression of the c-Ha-ras oncogene as compared to the parental cell line. These changes were not present in later cultures of MGH-U1/OCI cells, and no consistent difference in the levels of gene amplification or expression between the parent line and the sublines was found. Thus the content and expression of the activated c-Ha-ras oncogene does not correlate with malignant properties of the sublines.