scholarly journals CXCL12-Abundant Reticular (CAR) Cells Direct Megakaryocyte Protrusions Across the Bone Marrow Sinusoid Wall

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 722
Author(s):  
Nicole Wagner ◽  
Kristina Mott ◽  
Berin Upcin ◽  
David Stegner ◽  
Harald Schulze ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Blood Circulation ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Tissue Sections ◽  
Complex Process ◽  
Block Face ◽  
Platelet Release ◽  
Cell Retraction

Megakaryocytes (MKs) release platelets into the lumen of bone marrow (BM) sinusoids while remaining to reside within the BM. The morphogenetic events of this complex process are still not fully understood. We combined confocal laser scanning microscopy with transmission and serial block-face scanning electron microscopy followed by 3D-reconstruction on mouse BM tissue sections. These analyses revealed that MKs in close vicinity to BM sinusoid (BMS) wall first induce the lateral retraction of CXCL12-abundant reticular (CAR) cells (CAR), followed by basal lamina (BL) degradation enabling direct MK-sinusoidal endothelial cells (SECs) interaction. Subsequently, an endothelial engulfment starts that contains a large MK protrusion. Then, MK protrusions penetrate the SEC, transmigrate into the BMS lumen and form proplatelets that are in direct contact to the SEC surface. Furthermore, such processes are induced on several sites, as observed by 3D reconstructions. Our data demonstrate that MKs in interaction with CAR-cells actively induce BMS wall alterations, including CAR-cell retraction, BL degradation, and SEC engulfment containing a large MK protrusion. This results in SEC penetration enabling the migration of MK protrusion into the BMS lumen where proplatelets that are adherent to the luminal SEC surface are formed and contribute to platelet release into the blood circulation.

A New Approach to Phenotyping Disseminated Tumor Cells: Methodological advances and Clinical Implications

2000 ◽  
Vol 15 (1) ◽  
pp. 100-104 ◽  
Author(s):  
F. Noack ◽  
M. Schmitt ◽  
J. Bauer ◽  
D. Helmecke ◽  
W. Krüger ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Cells ◽  
Laser Scanning ◽  
Simultaneous Detection ◽  
Disseminated Tumor Cells ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Prognostic Impact ◽  
Primary Therapy ◽  
Double Immunofluorescence

At the time of primary therapy (surgery, systemic chemotherapy and/or radiation), disseminated tumor cells in the bone marrow can be found in almost one-third of patients with cancer of the breast, ovary, esophagus, stomach, colon, and other solid tumors. Whereas the prognostic impact of the mere presence of these cells is still a matter of debate, it has been shown that expression of tumor-associated antigens in disseminated tumor cells is linked to more aggressive disease. Therefore, further characterization of disseminated tumor cells at the protein and gene level has become increasingly important. To date, the most common detection method for disseminated tumor cells in the bone marrow is an immunocytochemical approach using cytokeratin-directed antibodies for detection of epithelial cells and the APAAP system for their visualization. We have established a new double immunofluorescence technique enabling simultaneous detection, phenotyping, and antigen quantification of disseminated tumor cells. Mononuclear cells from bone marrow are enriched by Ficoll gradient centrifugation and cytospins are prepared. Double immunofluorescence is performed using antibodies against cytokeratins 8/18/19 (mAb A45B/B3) and the uPA receptor CD87 (pAb HU277). CD87 expression is recorded by confocal laser scanning microscopy (CLSM) using fluorescence labeled latex beads as the reference; staining intensities of all the scans are then summed and quantified (extended focus). This protocol, originally designed for disseminated tumor cells in bone marrow, can also be applied to disseminated tumor cells in blood, to leukapheresis cells or to cells present in malignant ascites or other malignant effusions. The tumor cells detected may be used for gene and mRNA analyses. Furthermore, disseminated tumor cells also represent interesting targets for clinical studies on patient prognosis or prediction of therapy response as well as for specific tumor-biological therapies.

scholarly journals Implementation of Accurate and Fast DNA Cytometry by Confocal Microscopy in 3D

2005 ◽  
Vol 27 (4) ◽  
pp. 225-230
Author(s):  
Lennert S. Ploeger ◽  
André Huisman ◽  
Jurryt van der Gugten ◽  
Dionne M. van der Giezen ◽  
Jeroen A. M. Beliën ◽  
...  
Keyword(s):  
Genomic Instability ◽  
Confocal Laser Scanning Microscopy ◽  
Dna Content ◽  
Laser Scanning ◽  
Clinical Applications ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Dna Cytometry ◽  
Tissue Sections

Background: DNA cytometry is a powerful method for measuring genomic instability. Standard approaches that measure DNA content of isolated cells may induce selection bias and do not allow interpretation of genomic instability in the context of the tissue. Confocal Laser Scanning Microscopy (CLSM) provides the opportunity to perform 3D DNA content measurements on intact cells in thick histological sections. Because the technique is technically challenging and time consuming, only a small number of usually manually selected nuclei were analyzed in different studies, not allowing wide clinical evaluation. The aim of this study was to describe the conditions for accurate and fast 3D CLSM cytometry with a minimum of user interaction to arrive at sufficient throughput for pilot clinical applications. Methods: Nuclear DNA was stained in 14 μm thick tissue sections of normal liver and adrenal stained with either YOYO-1 iodide or TO-PRO-3 iodide. Different pre-treatment strategies were evaluated: boiling in citrate buffer (pH 6.0) followed by RNase application for 1 or 18 hours, or hydrolysis. The image stacks obtained with CLSM at microscope magnifications of ×40 or ×100 were analyzed off-line using in-house developed software for semi-automated 3D fluorescence quantitation. To avoid sectioned nuclei, the top and bottom of the stacks were identified from ZX and YZ projections. As a measure of histogram quality, the coefficient of variation (CV) of the diploid peak was assessed. Results: The lowest CV (10.3%) was achieved with a protocol without boiling, with 1 hour RNase treatment and TO-PRO-3 iodide staining, and a final image recording at ×60 or ×100 magnifications. A sample size of 300 nuclei was generally achievable. By filtering the set of automatically segmented nuclei based on volume, size and shape, followed by interactive removal of the few remaining faulty objects, a single measurement was completely analyzed in approximately 3 hours. Conclusions: The described methodology allows to obtain a largely unbiased sample of nuclei in thick tissue sections using 3D DNA cytometry by confocal laser scanning microscopy within an acceptable time frame for pilot clinical applications, and with a CV small enough to resolve smaller near diploid stemlines. This provides a suitable method for 3D DNA ploidy assessment of selected rare cells based on morphologic characteristics and of clinical samples that are too small to prepare adequate cell suspensions.

scholarly journals Elimination of Autofluorescence in Archival Formaldehyde-Fixed, Paraffin-Embedded Bone Marrow Biopsies

2018 ◽  
Vol 143 (3) ◽  
pp. 362-369 ◽  
Author(s):  
Wenxia Su ◽  
Lin Yang ◽  
Xueping Luo ◽  
Meng Chen ◽  
Jinqin Liu
Keyword(s):  
Bone Marrow ◽  
Efficient Method ◽  
Laser Scanning ◽  
Primary Myelofibrosis ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Immunofluorescence Analysis ◽  
Bone Marrow Biopsies ◽  
Hematologic Disorders ◽  
Paraffin Tissue

Context.— High levels of autofluorescence in bone marrow tissue constitute a major obstacle to immunofluorescence analysis of bone marrow biopsies. Objective.— To present a simple, efficient method to eliminate autofluorescence in bone marrow biopsies. Design.— Autofluorescence of paraffin bone marrow tissues was examined in different hematologic disorders with confocal laser scanning microscopy. Strong autofluorescence was observed in primary myelofibrosis and acute leukemia with reticulin myelofibrosis in 488-nm and 561-nm channels. To eliminate autofluorescence, AutoFluo Quencher was used on bone marrow sections with different incubation times. The effects of AutoFluo Quencher on immunofluorescence analysis of bone marrow biopsies was tested using antibodies tagged with different fluorophores. Results.— AutoFluo Quencher thoroughly eliminated the strong autofluorescence of bone marrow but did not decrease the intensity of fluorophores, leaving the specific signals of target proteins clearly visible. Conclusions.— This study presents a simple, efficient method to eliminate autofluorescence in bone marrow paraffin tissue, and it opens the way to better results in the immunofluorescence analysis of bone marrow biopsies.

scholarly journals Characterization and Quenching of Autofluorescence in Piglet Testis Tissue and Cells

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Yanfei Yang ◽  
Ali Honaramooz
Keyword(s):  
Laser Scanning ◽  
Intrinsic Fluorescence ◽  
Laser Scanning Microscopy ◽  
Epifluorescence Microscopy ◽  
Fluorescence Labeling ◽  
Confocal Laser ◽  
Target Cells ◽  
Tissue Sections ◽  
Sudan Black B ◽  
Testis Tissue

Significant intrinsic fluorescence in tissues and in disassociated cells can interfere with fluorescence identification of target cells. The objectives of the present study were (1) to examine an intrinsic fluorescence we observed in both the piglet testis tissue and cells and (2) to test an effective method to block the autofluorescence. We observed that a number of granules within the testis interstitial cells were inherently fluorescent, detectable using epifluorescence microscopy, confocal laser scanning microscopy, and flow cytometry. The emission wavelength of the autofluorescent substance ranged from 425 to 700 nm, a range sufficiently broad that could potentially interfere with fluorescence techniques. When we treated the samples with Sudan Black B for different incubation times, the intrinsic fluorescence was completely masked after treatment for 10–15 min of the testis tissue sections or for 8 min of the testis cells, without compromising specific fluorescence labeling of gonocytes with lectin Dolichos biflorus agglutinin (DBA). We speculate that the lipofuscin or lipofuscin-like pigments within Leydig cell granules were mainly responsible for the observed intrinsic fluorescence in piglet testes. The method described in the present study can facilitate the identification and characterization of piglet gonocytes using fluorescence microscopy.

scholarly journals Control of Autofluorescence of Archival Formaldehyde-fixed, Paraffin-embedded Tissue in Confocal Laser Scanning Microscopy (CLSM)

2001 ◽  
Vol 49 (12) ◽  
pp. 1565-1571 ◽  
Author(s):  
Werner Baschong ◽  
Rosmarie Suetterlin ◽  
R. Hubert Laeng
Keyword(s):  
Bone Marrow ◽  
Sodium Borohydride ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Frozen Sections ◽  
Paraffin Sections ◽  
Fixed Tissue ◽  
Tissue Processing ◽  
Sudan Black B

Confocal laser scanning microscopy (CLSM) offers the advantage of quasi-theoretical resolution due to absence of interference with out-of-focus light. Prerequisites include minimal tissue autofluorescence, either intrinsic or induced by fixation and tissue processing, and minimal background fluorescence due to nonspecific binding of the fluorescent label. To eliminate or reduce autofluorescence, three different reagents, ammonia-ethanol, sodium borohydride, and Sudan Black B were tested on paraffin sections of archival formaldehyde-fixed tissue. Paraffin sections of biopsy specimens of human bone marrow, myocardium, and of bovine cartilage were compared by CLSM at 488-nm, 568-nm and 647-nm wavelengths with bone marrow frozen sections fixed either with formaldehyde or with glutaraldehyde. Autofluorescence of untreated sections related to both the specific type of tissue and to the tissue processing technique, including fixation. The reagents' effects also depended on the type of tissue and technique of tissue processing, including fixation, and so did the efficiency of the reagents tested. Therefore, no general recipe for the control of autofluorescence could be delineated. Ammonia-ethanol proved most efficient in archival bone marrow sections. Sudan Black B performed best on myocardium, and the combination of all three reagents proved most efficient on paraffin sections of cartilage and on frozen sections fixed in formaldehyde or glutaraldehyde. Sodium borohydride was required for the reduction of unwanted fluorescence in glutaraldehyde-fixed tissue. In formaldehyde-fixed tissue, however, sodium borohydride induced brilliant autofluorescence in erythrocytes that otherwise remained inconspicuous. Ammonia-ethanol is believed to reduce autofluorescence by improving the extraction of fluorescent molecules and by inactivating pH-sensitive fluorochromes. The efficiency of borohydride is related to its capacity of reducing aldehyde and keto-groups, thus changing the fluorescence of tissue constituents and especially of glutaraldehyde-derived condensates. Sudan Black B is suggested to mask fluorescent tissue components.

scholarly journals Histochemical and immunocytochemical localization of prolactin receptors on Nb2 lymphoma cells: applications of confocal microscopy.

1990 ◽  
Vol 38 (7) ◽  
pp. 965-973 ◽  
Author(s):  
E Michel ◽  
J A Parsons
Keyword(s):  
Laser Scanning ◽  
Hormone Receptors ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Lymphoma Cells ◽  
Tissue Sections ◽  
Uv Illumination ◽  
Lactogenic Hormone ◽  
Lactogenic Hormones ◽  
Ovine Prolactin

We studied prolactin (PRL) binding sites on Nb2 lymphoma cells using two different light microscopic methods. First, histochemical detection was accomplished by using an aminomethyl coumarin-acetic acid-conjugated ovine prolactin molecule (AMCA-oPRL) on both glutaraldehyde-fixed and unfixed Nb2 lymphoma cells. Binding of AMCA-oPRL was studied after UV illumination and appeared as punctate fluorescence associated with many but not all cells. Binding was abolished when tissue sections were treated with excess unlabeled lactogenic hormones and was unchanged when a non-lactogenic hormone was used for displacement. Counting revealed significant differences between the number of labeled cells in populations known to exhibit up- or down-regulated PRL receptors. Second, indirect immunocytochemistry of Nb2 PRL receptors was accomplished by immunological detection of exogenously added ovine PRL using two antisera directed against ovine PRL. Visualization of the ligand-antibody complexes was accomplished by confocal laser scanning microscopy. Staining was restricted to a subpopulation of cells. The morphological results presented here add to the previous physiological and biochemical data on the presence of lactogenic hormone receptors on Nb2 lymphoma cells.

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