Wim Renmans

Potential of immunogold-silver staining for the study of leukocyte subpopulations as defined by monoclonal antibodies.

The potential of immunogold-silver staining for study of leukocyte subpopulations, as defined by monoclonal antibodies in cell suspensions, was examined. The cells were labeled in suspension as described for immunogold staining. Cytocentrifuge preparations of the suspensions were then immersed in a physical developer. By light microscopy, cells reacting with the monoclonal antibodies showed dark granules on their surface membrane. The morphology of the cells, as revealed by a panoptic counterstain, was comparable with that seen in routine cell smears for differential counts. The numbers of T-cells, T-helper/inducer cells, and T-cytotoxic/suppressor cells counted by this method in normal peripheral blood were nearly identical to those identified by immunogold staining and immunofluorescence microscopy in the same cell suspensions. The good morphological delineation also made possible rapid and accurate identification of particular leukocyte subsets in complex cell suspensions. Atypical lymphocytes from patients with infectious mononucleosis displayed the surface phenotype of activated T-cytotoxic/suppressor cells. Different maturation stages of neoplastic cells in patients with acute myeloid leukemia showed differences in surface antigen expression. Immunological detection of cell surface antigens could be combined with cytochemical staining of intracellular enzymatic activities. Finally, the labeling could be performed on cells prefixed on glass slides.

Sensitive detection of immunogold-silver staining with darkfield and epi-polarization microscopy.

We evaluated the contribution of darkfield and epi-polarization microscopy to the detection of leukocyte cell surface antigens with immunogold-silver staining (IGSS). Lymphocyte cell surface differentiation antigens were labeled with monoclonal antibodies and IGSS as described for brightfield microscopy. In darkfield and epi-polarization microscopy the labeling appeared as bright spots on a dark background. The sensitivity of detection was much higher than that of brightfield microscopy. Sixteenfold higher dilutions of the monoclonal antibody could be used to detect all cells expressing the antigen in the cell suspension. However, non-specific staining was also better visualized. The latter could be reduced to a level comparable to that of brightfield microscopy only by use of weaker labeling conditions. A 25% reduction of the silver enhancement time was necessary for this purpose. However, these weaker labeling conditions also reduced the intensity of the specific staining. Therefore, the efficiency of IGSS, as detected with darkfield and epi-polarization microscopy, was only fourfold greater than that found with brightfield microscopy or that of an immunofluorescence procedure. Especially in combination with transmitted light, to improve cell identification, epi-polarization microscopy is a reliable and sensitive method for detection of immunogold-silver-labeled cell surface antigens for diagnostic and research purposes.

Circulating CD34+ cells in cord blood and mobilized blood have a different profile of adhesion molecules than bone marrow CD34+ cells.

Abstract: The expression of adhesion molecules was studied on CD34+ hematopoietic precursors in cord blood, bone marrow and mobilized blood. The samples were labeled in a double immunofluorescence procedure with a CD34 monoclonal antibody and with antibodies against maturation and differentiation antigens and adhesion molecules. Myeloid precursors formed the majority of the CD34+ cells in all samples. In bone marrow a separate cluster of B‐cell precursors with low forward scatter was present. Nearly all CD34+ cells in normal bone marrow expressed VLA‐4 and VLA‐5, PECAM‐1, LFA‐3 and HCAM. The majority of the CD34+ cells also had LFA –1 and L‐selectin on the surface membrane. A small subset was VLA‐2, VLA‐3, ICAM‐1 or Mac‐1 positive. CD34+ cells expressing the vitronectin receptor or the CD11c antigen were rare. Cord blood and mobilized blood CD34+ cells had a lower expression of VLA‐2, VLA‐3 and VLA‐5 and a higher expression of LFA‐1, ICAM‐1 and L‐selectin than bone marrow CD34+ cells. Except for LFA‐1, this was not due to the presence of more myeloid precursors in these samples. Low β1 integrin expression may lead to less adhesion to the extracellular matrix. High expression of L‐selectin may facilitate interaction with endothelial cells. Therefore, this phenotype may favour mobilization.

An immunogold-silver staining method for detection of cell-surface antigens in light microscopy.

An immunogold-silver staining technique for detection of cell-surface antigens in cell suspensions was developed. Leukocyte cell suspensions were first incubated with monoclonal antibodies directed against cell-surface antigens and then with colloidal gold-labeled goat anti-mouse antibodies. Cytocentrifuge preparations of the cell suspensions were immersed in a physical developer containing silver lactate and hydroquinone as reducing substance. The preparations were then counterstained and mounted. In light microscopy, cells reacting with the monoclonal antibodies showed dark granules on their surface membrane. An optimal morphology, as revealed by a May-Grünwald-Giemsa counterstain, permitted accurate cell identification. The labeling was influenced by the gold particle diameter and the concentration of the gold reagents, by the duration of incubation in the physical developer, and by the composition and temperature of this medium. The T-cell subsets enumerated with this method in the peripheral blood of normal adults were identical to those found with other methods. The sensitivity of the technique was comparable with that of immunofluorescence microscopy. This immunogold-silver staining procedure proved to be a reliable tool for detection of cell-surface antigens in light microscopy.

Different expression of adhesion molecules on CD34+ cells in AML and B-lineage ALL and their normal bone marrow counterparts

Abstract:  The expression of adhesion molecules on CD34+ cells in acute myeloid leukemia (AML) and B‐lineage acute lymphoblastic leukemia (B‐lineage ALL) was compared with that on the myeloid and B‐lymphoid CD34+ cells in normal bone marrow. Bone marrow aspirates of 10 patients with AML, 8 patients with B‐lineage ALL and of 6 healthy volunteers were examined. The phenotype of the CD34+ cells was determined with a double immunofluorescence method and flow cytometry. CD34+ cells in AML and B‐lineage ALL showed a lower expression of VLA‐2 and VLA‐3 and a higher expression of ICAM‐1 and LFA‐3 than their normal bone marrow counterparts. AML CD34+ cells had less L‐selectin but more VLA‐5 on their surface membrane than normal myeloid CD34+ cells. B‐lineage ALL CD34+ cells showed an overexpression of LFA‐3. In individual patients deficiencies or over‐expression of the β1 integrin chain, VLA‐4, PECAM‐1 or HCAM also occurred. An abnormal adhesive capacity of the leukemic cells may influence their proliferation, their localisation and apoptosis. An aberrant expression of adhesion molecules may be used for the detection of minimal residual leukemia in these patients.

An immunogold-silver staining method for detection of cell surface antigens in cell smears.

We developed an indirect immunogold-silver staining method for detection of leukocyte cell surface antigens in cell smears. Air-dried and fixed cytocentrifuge preparations or smears of peripheral blood leukocytes were incubated with monoclonal antibodies (MAb) and colloidal gold-labeled secondary antibodies. The preparations were post-fixed and silver enhancement was performed. The smears were counterstained with May-Grunwald-Giemsa and examined in brightfield light microscopy. The morphology of the cells was well preserved. Leukocytes reacting with the MAb showed black granules on their surface membranes. The intense immunostaining and the low background allowed a rapid enumeration of the positive cells. The labeling could be detected with high sensitivity by epipolarization microscopy. This immunogold-silver staining method was used to quantify T- and B-lymphocytes and natural killer cells in buffy coat smears of normal adult blood. These lymphocyte subsets correlated well with those obtained in smears with the alkaline phosphatase-anti-alkaline phosphatase (APAAP) method and with those found by labeling of mononuclear cells in suspension with immunogold-silver staining. This immunogold-silver staining method forms a good alternative to immunoenzyme methods for study of hematologic cells. In addition, it could be a general procedure for detection of cell surface antigens in all kinds of cell smears.

Growth factor receptor profile of CD34+ cells in AML and B‐lineage ALL and in their normal bone marrow counterparts

Abstract: Leukaemic cells show a low clonogenic activity and a heterogeneous proliferative response to growth factors. We investigated whether this could be due to an altered expression of growth factor receptors on the leukaemic precursors. Receptors for G‐CSF, stem cell factor (SCF), IL‐3, IL‐6 and IL‐7 were detected on CD34+ cells in AML and B‐lineage ALL with monoclonal antibodies and flow cytometry. The expression was compared with that on myeloid and B‐lymphoid CD34+ cells in normal bone marrow. Leukaemic CD34+ cells expressed the same receptors as their normal counterparts. AML and B‐lineage ALL could be distinguished by the growth factor receptor profile of their CD34+ cells. SCFR, G‐CSFR and IL‐6Rα were found in AML, IL‐7R in B‐lineage ALL and IL‐3Rα in both. IL‐3Rα was upregulated in AML and B‐lineage ALL CD34+ cells, while samples with low or high expression were present for the other receptors. This variable expression could correlate with the heterogeneous response of leukaemic cells to growth factors. Functional studies on isolated CD34+ cells are needed to investigate this further.

Preclinical evaluation of invariant natural killer T cells in the 5T33 multiple myeloma model.

Immunomodulators have been used in recent years to reactivate host anti-tumor immunity in several hematological malignancies. This report describes the effect of activating natural killer T (NKT) cells by α-Galactosylceramide (α-GalCer) in the 5T33MM model of multiple myeloma (MM). NKT cells are T lymphocytes, co-expressing T and NK receptors, while invariant NKT cells (iNKTs) also express a unique semi-invariant TCR α-chain. We followed iNKT numbers during the development of the disease in both 5T33MM mice and MM patients and found that their numbers dropped dramatically at the end stage of the disease, leading to a loss of total IFN-γ secretion. We furthermore observed that α-GalCer treatment significantly increased the survival of 5T33MM diseased mice. Taken together, our data demonstrate for the first time the possibility of using a preclinical murine MM model to study the effects of α-GalCer and show promising results of α-GalCer treatment in a low tumor burden setting.

Effects of Physical Exercise on Markers of Cellular Immunosenescence: A Systematic Review

Aging affects negatively the immune system, defined as immunosenescence, which increases the susceptibility of elderly persons to infection, autoimmune disease, and cancer. There are strong indications that physical exercise in elderly persons may prevent the age-related decline in immune response without significant side effects. Consequently, exercise is being considered as a safe mode of intervention to reduce immunosenescence. The aim of this review was to appraise the existing evidence regarding the impact of exercise on surface markers of cellular immunosenescence in either young and old humans or animals. PubMed and Web of Science were systematically screened, and 28 relevant articles in humans or animals were retrieved. Most of the intervention studies demonstrated that an acute bout of exercise induced increases in senescent, naïve, memory CD4+ and CD8+ T-lymphocytes and significantly elevated apoptotic lymphocytes in peripheral blood. As regards long-term effects, exercise induced increased levels of T-lymphocytes expressing CD28+ in both young and elderly subjects. Few studies found an increase in natural killer cell activity following a period of training. We can conclude that exercise has considerable effects on markers of cellular aspects of the immune system. However, very few studies have been conducted so far to investigate the effects of exercise on markers of cellular immunosenescence in elderly persons. Implications for immunosenescence need further investigation.

Shortcomings in the Application of Multicolour Flow Cytometry in Lymphocyte Subsets Enumeration

The lymphoid system is composed of numerous phenotypically distinct subsets of cells, each of which has a unique role in the effectiveness of an immune response. To distinguish specifically between these subsets, it is mandatory to detect simultaneously different cell surface antigens. This became feasible by the development of multicolour flow cytometric technologies. With these techniques, researchers now have the opportunity to study individual cells in far greater detail than previously possible. However, proper data analysis, interpretation and presentation of results will require a high level of understanding of the intricacies of the technology and the inherent limitations of the acquired data. The present report is intended to contribute to the better understanding of how the flow cytometer operates. This report may help new and inexperienced users to work appropriately with the flow cytometer.