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H J Tanke

Publications and source records attributed to H J Tanke.

At least 19 recordsLinked to original sources
PubMed1992

Quantitation of polymerase chain reaction products by hybridization-based assays with fluorescent, colorimetric, or chemiluminescent detection.

In this report two nonradioactive assays for quantitative analysis of polymerase chain reaction (PCR) products are presented. In the first assay, magnetic beads coated with streptavidin were used to capture biotinylated PCR fragments. After hybridization with a hapten-labeled probe, these beads were analyzed either by flow cytometry (method A) or by immunoenzymatic reactions (method B). Using a dilution series of purified PCR products, we consistently found a lower detection limit of 1.5 fmol for method A than the 0.15-fmol limit for method B. In the second assay we used the peroxidase-based enhanced chemiluminescence system in combination with a cooled charge-coupled device camera to quantify PCR fragments that were spotted on membranes. A linear logarithmic response was observed between the amount of light produced within a certain time interval and the number of DNA molecules. With an exposure time of 5 min, a detection limit of 0.15 fmol was found. Longer exposure times did not result in a higher sensitivity. We conclude that the assays are of sufficient sensitivity for application in quantitative PCR strategies. The nonradioactive technology facilitates implementation of these assays in routine settings.

Bacterial Proteins↗
PubMed1992

Preparation and microscopic visualization of multicolor luminescent immunophosphors.

The preparation of charge-stabilized suspensions of small phosphor particles (0.1-0.3 micron) and their coupling with antibodies to immunoreactive conjugates is described. Phosphor particles consisting of yttriumoxisulfide activated with europium served as a model system in the evaluation of the stabilizing properties of several polycarboxylic acids. The optimal reagents were then applied to other phosphors which differ in spectral characteristics as well as in luminescence lifetime. These phosphors were ground to a size of 0.1-0.3 micron and proteins or other macromolecules were adsorbed to the phosphor particles to prepare conjugates of different physico-chemical properties. A time-resolved microscope, suitable for real time visualization of the time-delayed luminescence of the immunophosphors by the human eye, is described in detail. Since most phosphors require excitation with far UV light, a special fluorescence microscope allowing far UV excitation was developed for conventional visualization of the luminescence emitted by the phosphor. The possibility of multiple color labeling using various phosphor conjugates was demonstrated in a model system consisting of haptenized latex beads.

Adsorption↗
PubMed1992

Quantification of inter- and intra-nuclear variation of fluorescence in situ hybridization signals.

This study aims at the quantification of specific DNA sequences by using fluorescence in situ hybridization (ISH) and digital imaging microscopy. The cytochemical and cytometric aspects of a quantitative ISH procedure were investigated, using human peripheral blood lymphocyte interphase nuclei and probes detecting high copy number target sequences as a model system. These chromosome-specific probes were labeled with biotin, digoxigenin, or fluorescein. Quantification of the fluorescence ISH signals was performed using an epifluorescence microscope equipped with a multi-wavelength illuminator, and a cooled charge coupled device (CCD) camera. Specific image analysis programs were developed for the segmentation and analysis of the images provided by ISH. The fluorescence intensity distributions of the ISH spots showed large internuclear variation (CVs up to 65%) for the probes used. The variation in intensity was found to be independent of the probe, the type of labeling, and the type of immunocytochemical detection used. Variation in intensity was not caused primarily by the immunocytochemical detection method, since directly fluorescein-labeled probes showed similar internuclear variation. Furthermore, it was found that different white blood cell types, which harbor different degrees of compactness of the nuclear chromatin, showed the same variation. The intra-nuclear variation in intensity of the ISH spots on the two chromosome homologs within one nucleus was significantly smaller (approximately 20%) than the inter-nuclear variation, probably due to more constant local hybridization conditions. Due to the relatively small intranuclear variation, copy number polymorphisms of the satellite DNA sequence on chromosome 1 could readily be quantified.(ABSTRACT TRUNCATED AT 250 WORDS)

Analog-Digital Conversion↗
PubMed1992

Fluorescence ratio measurements of double-labeled probes for multiple in situ hybridization by digital imaging microscopy.

To expand the multiplicity of the in situ hybridization (ISH) procedure, which is presently limited by the number of fluorochromes spectrally separable in the microscope, a digital fluorescence ratio method is proposed. For this purpose, chromosome-specific repetitive probes were double-labeled with two haptens and hybridized to interphase nuclei of human peripheral blood lymphocytes. The haptens were immunocytochemically detected with specific antibodies conjugated with the fluorochromes FITC or TRITC. The FITC and TRITC fluorescence intensities of spots obtained with different double-haptenized probes were measured, and the fluorescence ratio was calculated for each ISH spot. Combinations of different haptens, such as biotin, digoxigenin, fluorescein, sulfonate, acetyl amino fluorene (AAF), and mercury (Hg) were used. The fluorescence intensity ratio (FITC/TRITC) of the ISH spots was fairly constant for all combinations used, with coefficients of variation between 10 and 30%. To study the feasibility of a probe identification procedure on the basis of probe hapten ratios, one probe was double-labeled with different ratios, by varying the relative concentrations of the modified nucleotides (biotin-11-dUTP and digoxigenin-11-dUTP) in the nick-translation reaction. Measurement of the FITC and TRITC intensities of the ISH spots showed that the concentration of modified nucleotides used in the labeling procedures was reflected in the mean fluorescence intensity of the ISH spots. Furthermore, the ratio distributions showed little overlap due to the relatively small coefficients of variation. The results indicate that a multiple ISH procedure based on fluorescence ratio imaging of double-labeled probes is feasible.

Analog-Digital Conversion↗
PubMed1992

Quantification of fluorescence in situ hybridization signals by image cytometry.

In this study we aimed at the development of a cytometric system for quantification of specific DNA sequences using fluorescence in situ hybridization (ISH) and digital imaging microscopy. The cytochemical and cytometric aspects of a quantitative ISH procedure were investigated, using human peripheral blood lymphocyte interphase nuclei and probes detecting high copy number target sequences as a model system. These chromosome-specific probes were labeled with biotin, digoxigenin, or fluorescein. The instrumentation requirements are evaluated. Quantification of the fluorescence ISH signals was performed using an epi-fluorescence microscope with a multi-wavelength illuminator, equipped with a cooled charge couple device (CCD) camera. The performance of the system was evaluated using fluorescing beads and a homogeneously fluorescing specimen. Specific image analysis programs were developed for the automated segmentation and analysis of the images provided by ISH. Non-uniform background fluorescence of the nuclei introduces problems in the image analysis segmentation procedures. Different procedures were tested. Up to 95% of the hybridization signals could be correctly segmented using digital filtering techniques (min-max filter) to estimate local background intensities. The choice of the objective lens used for the collection of images was found to be extremely important. High magnification objectives with high numerical aperture, which are frequently used for visualization of fluorescence, are not optimal, since they do not have a sufficient depth of field. The system described was used for quantification of ISH signals and allowed accurate measurement of fluorescence spot intensities, as well as of fluorescence ratios obtained with double-labeled probes.

Analog-Digital Conversion↗
PubMed1992

Immunochemical detection of proteins and nucleic acids on filters using small luminescent inorganic crystals as markers.

A new luminescent marker for the immunochemical detection of proteins and nucleic acids on filters is reported. The label consists of inorganic crystals, generally called phosphors, with a particle size of 0.1-0.3 microns, stabilized in suspension with polycarboxylic acids and subsequently conjugated to immunoreactive macromolecules. Immunophosphor conjugates exhibit slowly decaying fluorescence that is strong and practically nonfading and not sensitive to quenching by water molecules. They are therefore suited for conventional fluorescence detection as well as for time-resolved detection. The lifetime of the phosphors was in the micro/milliseconds range upon excitation with ultraviolet light. Proteins or nucleic acids immobilized on nitrocellulose filters were detected immunochemically or by hybridization, using haptenized nucleic acid probes followed by immunochemical detection, respectively. The ultimate detection limit of proteins, using phosphor-labeled macromolecules including an immunochemical amplification step, was found to be 10 fg. The detection limit of nucleic acids was 300 fg for demonstration of hapten-labeled probes and 10 pg in hybridization formats with hapten-labeled probes. The sensitivity of methods using phosphor-labeled macromolecules was in all cases as good as or better than that of methods using alkaline phosphatase developed to NBT/BCIP. The use of immunophosphors for detection of proteins and nucleic acids on Western and Southern blots is demonstrated. Finally, the use of multiple phosphors with different kinetic and spectral characteristics for multiparameter studies is indicated.

Alkaline Phosphatase↗
PubMed1992

Quantification of sensitive non-isotopic filter hybridizations using the peroxidase catalyzed luminol reaction.

The development of a sensitive, non-isotopic filter hybridization method based on the peroxidase catalyzed luminol reaction is described. High sensitivity was achieved by optimizing the conditions of the hybridization procedure, the immunochemical detection and the peroxidase/luminol reaction. This resulted in the reproducible detection of 10-30 femtogram of target DNA on blots within minutes when a cooled charge coupled device (CCD) camera was used to record the luminescence signal. After optimalization, the system was successfully applied for the qualitative and quantitative analysis of small amounts of DNA in dot-blots as well as in Southern blot analysis.

Biotin↗
PubMed1991

In situ hybridization with fluoresceinated DNA.

We have used fluorescein-11-dUTP in a nick-translation format to produce fluoresceinated human nucleic acid probes. After in situ hybridization of fluoresceinated DNAs to human metaphase chromosomes, the detection sensitivity was found to be 50-100 kb. The feasibility and the increase in detection sensitivity of microscopic imaging of in situ hybridized, fluoresceinated DNA with an integrating solid state camera for rapid cosmid mapping is illustrated. Combination of fluoresceinated DNA with biotinated and digoxigeninated DNAs allowed easy performance of triple fluorescence in situ hybridization. The potential of these techniques for DNA mapping, cytogenetics and biological dosimetry is briefly discussed.

DNA Probes↗
PubMed1991

Flow cytometric double labeling technique for screening of multidrug resistance.

We investigated the capabilities of flow cytometry in the analysis of a multidrug resistant (MDR) human ovarian cancer cell line 2780AD and its drug sensitive parental A2780. A functional assay using daunorubicin (DNR) as a fluorescent probe was combined with an immunofluorescence assay of P-glycoprotein (P-gp) using the monoclonal antibody MRK-16. Functionally MDR could be demonstrated by the lower DNR-content of MDR cells compared to DNR-content of drug sensitive cells. When incubation was performed with DNR in the presence of verapamil, DNR-content increased in the MDR cells. However the content of the A2780 cells was never attained. Differences in DNR-content were not related to differences in DNA-content. In experimental cell lines immunofluorescence data were inversely related with those of DNR-content: MDR cells had high levels of P-gp expression and low levels of DNR-content (and vice versa in drug sensitive cells). Both assays can be easily combined in a multiparametric flow cytometric procedure to evaluate both parameters simultaneously in the same cells. Analysis of clinical samples demonstrates the existence of aberrant subpopulations which would not be detected by using a single parameter assay.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Detection of mixed chimaerism in flow-sorted cell subpopulations by PCR-amplified VNTR markers after allogeneic bone marrow transplantation.

The amplification of Variable Number of Tandem Repeats (VNTR) by the polymerase chain reaction (PCR) was used to determine the extent of chimaerism in flow sorted lymphoid and myeloid cell populations following allogeneic bone marrow transplantation (BMT). Pre-BMT screening with a set of five VNTR revealed that at least one marker was maximally informative in 95% of donor-recipient pairs. Mixing reconstruction experiments indicated that detection of 1-5% of the minor cell population in a sample of 5 x 10(3) nucleated cells is feasible. Flow sorted post-transplant peripheral blood B- and T-lymphocyte, natural killer and monocyte cell populations were subjected to PCR-VNTR marker analysis. It was shown that this procedure can be used for the early detection of engraftment and the identification of mixed chimaerism in various haematopoietic cell lineages in patients with leukaemia or severe combined immune deficiency, treated with allogeneic BMT.

Base Sequence↗
PubMed1990

Gating of the so-called 'lymphocytic' cell population for the quantification of natural killer cells (CD16+) by flow cytometry causes loss of CD16 positive cells.

Natural killer cells can phenotypically be identified as CD16 positive with a specific monoclonal antibody (B73.1 = Leu-11c) by either immunofluorescence microscopy or by flow cytometry. The standard procedure in flow cytometry is to set a window or gate around the so called lymphocytic population, based on scatter characteristics. In this paper we demonstrate that a substantial part of the NK cell population is situated outside this gate in the total mononuclear cell population. We therefore recommend that the number of CD16+ cells is determined in the total mononuclear cell population. However, in the total mononuclear cell population, a group of dimly CD16 positive cells, probably monocytes, interferes with a clear separation of cells with a positive and negative fluorescence. We describe two methods to overcome this problem.

Adult↗
PubMed1990

Multiple fluorescence in situ hybridization.

A method for multiple fluorescence in situ hybridization is described allowing the simultaneous detection of more than three target sequences with only three fluorescent dyes (FITC, TRITC, AMCA), respectively emitting in the green, red, and blue. This procedure is based on the labeling of (DNA) probes with more than one hapten and visualisation in multiple colors. The possibility to detect multiple targets simultaneously is important for prenatal diagnosis and the detection of numerical and/or structural chromosome aberrations in tumor diagnosis. It may form the basis for an in situ hybridization based chromosome banding technique.

2-Acetylaminofluorene↗
PubMed1990

Inorganic phosphors as new luminescent labels for immunocytochemistry and time-resolved microscopy.

A new strongly luminescent marker consisting of inorganic crystals is described for time-resolved microscopy. These crystals, known as phosphors, show delayed luminescence, unlike prompt fluorescent labels such as FITC, TRITC and phycobiliproteins, and are therefore potentially suitable for time-resolved microscopy. The luminescence of these phosphors is strong and non-fading in comparison to FITC/TRITC, and not significantly influenced by pH or temperature. The phosphor yttriumoxisulfide activated with europium emits maximally at 620 nm with a typical half life-time of approximately 700 musec, upon excitation with near ultraviolet light (360 nm). Phosphors for immunocytochemical staining were made by ball milling and were stabilized in suspension with polycarboxylic acids. Proteins such as avidin, protein A or immunoglobulins were allowed to adsorb to the surface of the phosphors. The immunocytochemical properties of the conjugates were evaluated in a model system of latex beads with defined surface antigens and in a cellular system containing fixed human lymphocytes or erythrocytes. Specific cytochemical staining was observed in suspension as well as on glass slides. A specially constructed time-resolved microscope was used to suppress the fast decaying fluorescence, thereby permitting visualization of the specific, slowly decaying luminescence of the phosphor label without the necessity of integration. Finally, the use of multiple phosphors with different kinetic and spectral characteristics for multiparameter studies is indicated.

Immunohistochemistry↗
PubMed1990

Adenosine deaminase complexing protein (ADCP) expression and metastatic potential in prostatic adenocarcinomas.

The expression of the adenosine deaminase complexing protein (ADCP) was investigated by immunohistochemistry in the normal and hyperplastic human prostate, in 30 prostatic adenocarcinomas, and in seven human prostatic adenocarcinoma cell lines grown as xenografts in athymic nude mice. In the normal and hyperplastic prostate, ADCP was localized exclusively in the apical membrane and the apical cytoplasm of the glandular epithelial cells. In prostatic adenocarcinomas, four distinct ADCP expression patterns were observed: diffuse cytoplasmic, membranous, both cytoplasmic and membranous, and no ADCP expression. The expression patterns were compared with the presence of metastases. We found an inverse correlation between membranous ADCP immunoreactivity and metastatic propensity. Exclusively membranous ADCP immunoreactivity occurred only in non-metastatic tumours. In contrast, the metastatic tumours showed no or diffuse cytoplasmic ADCP immunoreactivity. This suggests that immunohistochemical detection of ADCP might predict the biological behaviour of prostatic cancer. However, the occurrence of membranous ADCP immunoreactivity in the xenograft of a cell line (PC-EW), derived from a prostatic carcinoma metastasis, indicates that not only the tendency to metastasize modulates ADCP expression.

Adenocarcinoma↗
PubMed1990

Automated flow cytometric analysis of drug susceptibility of malaria parasites.

A method is described for the fully automated reading of Plasmodium falciparum drug susceptibility tests. Cultured material was fixed and could be stored for greater than or equal to 6 months until analysis. The parasites were stained for DNA with the fluorescent dye Hoechst 33258 and analyzed by flow cytometry. The procedure was done in 96-well microtiter plates, after which the material was directed through the sensing region in the flow cytometer. The data resulting from the analysis were stored by microcomputer and processed by a program developed for this purpose. Using this method, a number of different parameters describing the growth in culture can be assessed.

Animals↗
PubMed1989

Detection of chromosome aberrations in interphase tumor nuclei by nonradioactive in situ hybridization.

In a blind study, chromosome aberrations in tumor cells were analyzed by conventional cytogenetic techniques (G banding) and nonradioactive in situ hybridization with chromosome-specific probes. The material was obtained directly from patients with hematologic diseases and from colon tumor derived cell lines. The cytogenetic data obtained with G banding were in accord with those obtained by in situ hybridization to metaphase chromosomes. Most importantly, in situ hybridization to interphase nuclei gave reliable results and even allowed detection of cell subpopulations that were not detected by analyzing metaphase chromosomes. Furthermore, in retrospect, even structural aberrations could be detected in interphase nuclei; abnormal cells with either an i(1q) or a translocation der(1)t(1;7) could be identified. Our results show that the application of in situ hybridization in combination with routine cytogenetic techniques offers significant advantages for cytogenetic analysis of solid tumors and hematologic malignancies.

Adenocarcinoma, Mucinous↗