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 antileukocyte 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

Immuno-Cytochemistry with Backscattered Electrons

Some cytochemical reaction products are visible inside the cytoplasm of cells observed with the scanning electron microscope (SEM) using the backscattered electron imaging (BEI) mode. Methods can be utilized whenever they result in the deposition of heavy metal, like silver, lead or osmium at the sites of the enzymatic reaction. More recently the BEI mode of the SEM has been demonstrated to improve the detection of immunogold labeled cell surface antigens. Colloidal gold particles, 40 to 15 nm in diameter can be efficiently used for immuno-specific labeling. Moreover, cytochemical reactions can be applied to previously immunogold labeled cells, therefore combining the results of enzyme cytochemistry and of surface labeling at the level of each individual cell. The choice of fixative, incubation media, dehydration and drying methods should be guided by considerations on the sample characteristics for optimal electron scattering. Cytochemical as well as immuno-labeling reactions are not used per se but in combination with the study of cell surface morphology which needs, therefore, to be sufficiently well preserved. Coating should provide good conductivity and secondary electron emission, while emitting a minimal number of backscattered electrons. The application of these methods considerably enhances our capacity to characterize with the SEM the surface morphology of precisely identified subpopulations of many cell types

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

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

The prevalence and clinical relevance of p27 and cyclin E immunoreactivity in myelodysplastic syndromes

We report on p27 and cyclin E immunoreactivity (IR) in 86 patients with myelodysplastic syndromes (MDS). No association was found between cyclin E IR and survival in the whole series. Cyclin E IR was more prevalent in patients with refractory anemia (RA) and RA with ringed sideroblasts (RARS) with poor survival, although this trend was not statistically significant

Reduced intensity conditioning allogeneic transplant for advanced chronic lymphocytic leukemia

We report the preliminary results of 12 patients with advanced stage chronic lymphocytic leukemia (CLL) transplanted following reduced intensity conditioning (RIC. With a median of 22 months of follow-up, 9 patients are alive and 3 have died of progressive disease, graft-versus-host disease (GVHD) or toxic hepatitis. Acute grade I-III GVHD occurred in 33% of patients and chronic GVHD in 50%. Eight of the 12 patients achieved a complete remission (CR) and 2 patients a partial remission (PR). Donor lymphocyte infusion was effective in 6 patients. Event-free survival, progression-free survival and non-relapse mortality at 3 years were 68%, 42% and 16%, respectively. Our results show successful immunomodulation and reduction in tumor burden in high risk CL

Noninvasive near-infrared live imaging of human adult mesenchymal stem cells transplanted in a rodent model of Parkinson’s disease

Background: We have previously shown that human mesenchymal stem cells (hMSCs) can reduce toxin-induced neurodegeneration in a well characterized rodent model of Parkinson's disease. However, the precise mechanisms, optimal cell concentration required for neuroprotection, and detailed cell tracking need to be defined. We exploited a near-infrared imaging platform to perform noninvasive tracing following transplantation of tagged hMSCs in live parkinsonian rats.Methods: hMSCs were labeled both with a membrane intercalating dye, emitting in the near-infrared 815 nm spectrum, and the nuclear counterstain, Hoechst 33258. Effects of near-infrared dye on cell metabolism and proliferation were extensively evaluated in vitro. Tagged hMSCs were then administered to parkinsonian rats bearing a 6-hydroxydopamine-induced lesion of the nigrostriatal pathway, via two alternative routes, ie, intrastriatal or intranasal, and the cells were tracked in vivo and ex vivo using near-infrared technology.Results: In vitro, NIR815 staining was stable in long-term hMSC cultures and did not interfere with cell metabolism or proliferation. A significant near-infrared signal was detectable in vivo, confined around the injection site for up to 14 days after intrastriatal transplantation. Conversely, following intranasal delivery, a strong near-infrared signal was immediately visible, but rapidly faded and was completely lost within 1 hour. After sacrifice, imaging data were confirmed by presence/absence of the Hoechst signal ex vivo in coronal brain sections. Semiquantitative analysis and precise localization of transplanted hMSCs were further performed ex vivo using near-infrared imaging.Conclusion: Near-infrared technology allowed longitudinal detection of fluorescent-tagged cells in living animals giving immediate information on how different delivery routes affect cell distribution in the brain. Near-infrared imaging represents a valuable tool to evaluate multiple outcomes of transplanted cells, including their survival, localization, and migration over time within the host brain. This procedure considerably reduces the number of animal experiments needed, as well as interindividual variability, and may favor the development of efficient therapeutic strategies promptly applicable to patients

A pilot study of low-dose subcutaneous alemtuzumab therapy for patients with hemotherapy-refractory chronic lymphocytic leukemia

Subcutaneous low-dose alemtuzumab (10 mg t.i.w. for 18 weeks) induced a 50% response rate, including 25% complete response, in 16 patients with refractory chronic lymphocytic leukemia (CLL) patients. The responses were substantial even in patients with unfavorable cytogenetics, fludarabine/rituximab refractoriness, Rai stage IV, previous infections, and age over 65 years. Subcutaneous low-dose alemtuzumab is effective in poor prognosis B-CLL, and has a particularly favourable toxicity profile

RAS mutations vontribute to evolution of chronic myelomonocytic leukemia to the proliferative variant

Purpose: The biological and clinical heterogeneity of chronic myelomonocytic leukemia features renders its classification difficult. Moreover, because of the limited knowledge of the mechanisms involved in malignant evolution, chronic myelomonocytic leukemia remains a diagnostic and therapeutic challenge and a poor prognosis disease. We aimed to verify the biological and clinical significance of the discrimination, based on the leukocyte count, between myelodysplastic chronic myelomonocytic leukemia (MD-CMML) and myeloproliferative chronic myelomonocytic leukemia (MP-CMML). Experimental Design: Peripheral blood samples from 22 patients classified as MD-CMML and 18 as MP-CMML were collected at different time points during disease course, and patients' clinical characteristics were examined. RAS mutational screening was done by sequencing and, for each substitution identified, a highly selective allele-specific PCR was set up to screen all specimens. Results: MP-CMML patients showed a significantly poorer survival (P = 0.003) and a higher frequency of RAS mutations (P = 0.033) by sequencing compared with MD-CMML. Overall, five MD-CMML patients progressed to myeloproliferative disease: in two, allele-specific PCR unveiled low levels of the RAS mutations predominating in the myeloproliferative phase at the time of myelodysplastic disease, documenting for the first time the expansion of a RAS mutated clone in concomitance with chronic myelomonocytic leukemia evolution. Moreover, one of the progressed patients harbored the FLT3-ITD and two MP-CMML patients presented with the JAK2 V617F substitution. All these lesions were mutually exclusive. Conclusions: Our results strongly suggest RAS mutations to function as a secondary event that contributes to development of the chronic myelomonocytic leukemia variant with the poorer prognosis (MP-CMML) and therefore advise their detection to be implemented in chronic myelomonocytic leukemia diagnostics and monitorin