Chromosome specific DNA hybridization in suspension for flow cytometric detection of chimerism in bone marrow transplantation and leukemia

Flow cytometry was used to measure the fluorescence intensity of nuclei that were subjected to fluorescent in situ hybridization in suspension with chromosome specific DNA probes. Paraformaldehyde-fixed nuclei were protein digested with trypsin and hybridized simultaneously with a biotin-and DIG labeled chromosome specific centromere probe. A number of probes were tested in the suspension hybridizations. The method yielded fluorescent hybridization signals that allow discrimination between Y chromosome positive and negative nuclei when analyzed by flow cytometry. The method is especially suited for analysis of bone marrow cells derived from patients who have received a sex-mismatched allogeneic bone marrow transplantation. Male leukemia cells with a trisomy for chromosome 8 were mixed with normal female cells and simultaneously hybridized in suspension with a DIG labeled probe specific for chromosome 8 and the biotin labeled Y chromosome probe. Y chromosome positive or negative nuclei were s

The use of FISH with chromosome-specific repetitive DNA probes for the follow-up of leukemia patients

The use of fluorescence in situ hybridization (FISH) for the purpose of repeated follow-up examination of bone marrow samples from 38 leukemia patients was investigated. On the basis of conventional cytogenetic analysis, patients with acute leukemia whose leukemic cells carried numerical chromosomal aberrations were selected and followed with repetitive DNA probes that specifically hybridize to one chromosome type. Repeated cytogenetic metaphase analyses would have been laborious and not sensitive or quantitative enough to follow declining numbers of aberrant cells. FISH, as an interphase cytogenetic technique, provides a rapid and simple alternative with high sensitivity. Although FISH data before and after chemotherapy were in agreement with bone marrow cytology in 30 of 38 patients, discrepancies were noticed in specific cases. These could be explained by the presence of cytogenetically distinct subclones that behave differently during treatment, the presence of differentiated leukemic cells, changes in the chromosomal constitution caused by clonal relapse, or the fact that a numerical aberration is found by conventional chromosome banding analysis while the target region to which the probe is directed is still present in the nucleus as a diploid set

Orally administered 5-aminolevulinic acid for isolation and characterization of circulating tumor-derived extracellular vesicles in glioblastoma patients

Background: In glioblastoma (GB), tissue is required for accurate diagnosis and subtyping. Tissue can be obtained through resection or (stereotactic) biopsy, but these invasive procedures provide risks for patients. Extracellular vesicles (EVs) are small, cell-derived vesicles that contain miRNAs, proteins, and lipids, and possible candidates for liquid biopsies. GB-derived EVs can be found in the blood of patients, but it is difficult to distinguish them from circulating non-tumor EVs. 5-aminolevulinic acid (5-ALA) is orally administered to GB patients to facilitate tumor visualization and maximal resection, as it is metabolized to fluorescent protoporphyrin IX (PpIX) that accumulates in glioma cell

Flow karyotyping and fluorescence in situ hybridization

Cytogenetic analysis of tumors has become increasingly important in relation to diagnois, biologica

Identification and monitoring of effector and regulatory T cells during experimental arthritis based on differential expression of CD25 and CD134

Major problems in the analysis of CD4+effector cell and regulatory T cell (Treg) populations in an activated immune system are caused by the facts that both cell types can express CD25 and that the discriminatory marker forkhead box p3 can only be analyzed in nonviable (permeabilized) cells. Here, we show that CD134 (OX40) can be used as a discriminatory marker combined with CD25 to isolate and characterize viable CD4+effector cells and Tregs. Before and during adjuvant arthritis in rats, coexpression of CD134 and CD25 identified activated Tregs consistently, as these T cells proliferated poorly to disease-associated antigens and were suppressive in vitro and in vivo. Depending on the time of isolation and location, CD4+T cell populations expressing CD134 or CD25 contained effector/memory T cells. Analysis of the function, phenotype, and amount of the CD4+T cell subsets in different lymph node stations revealed spatiotemporal differences in effector cell and Treg compartments during experimental arthritis