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C Cremer

Publications and source records attributed to C Cremer.

At least 55 records · Page 3Linked to original sources
PubMed1995

Continuous focusing of biological particles by continuous immuno magnetic sorter: technique and applications.

We have developed a device for continuous deviation-mode, open-gradient fractionation of strongly magnetizable particles based on an electrophoresis counter-flow chamber. A mixture of magnetically labeled and nonlabeled particles can be injected into a given continuously flowing chamber buffer. The particles pass the inhomogeneous magnetic field of the open-gradient electromagnet in two narrow streams. According to the magnetic moments, induced by the magnetic field, magnetically labeled particles are deviated. The nonlabeled particles pass the magnetic field with negligible interaction. The deviated particles are focused into a stream that is completely separated from the streams of the nondeviated particles. The streams are fractionated by the counter-flow technique and collected in different vials. A high sorting purity, depending only on the specificity of the antibodies or similar labeling techniques, and a high through-put rate of up to 10(9) particles per hour was achieved. This was experimentally shown both by test particles and blood cells. The vital conditions of these blood cells were maintained by magnetic sorting.

B-Lymphocytes↗
PubMed1995

alpha-MSH-induced behavior: changes after diazepam and baclofen administration related with cyclic AMP levels.

The present work was performed to evaluate the participation of the benzodiacepinic GABAA and GABAB components upon excessive grooming, locomotion, rearing, and stretching/yawning syndrome induced by the intracerebroventricularly alpha-MSH administration by using GABAA and GABAB agonists. It also aims at evaluating possible relation between changes in cAMP levels in caudate-putamen and accumbens nuclei and the behavioral responses. Injection of diazepam or baclofen reduced the total behavioral scores in a dose-related manner as well as the cAMP levels with respect to the control values (animals treated with artificial cerebrospinal fluid). When diazepam was tested in animals simultaneously injected with alpha-MSH, behavioral scores decreased with respect to those treated with the peptide alone. Cyclic AMP also decreased after combined treatment (MSH + diazepam).

Animals↗
PubMed1994

Rapid fluorescence in situ hybridization with repetitive DNA probes: quantification by digital image analysis.

Fluorescence in situ hybridization (FISH) has become an important tool not only in cytogenetic research but also in routine clinical chromosome diagnostics. Here, results of a quantification of fluorescence signals after in situ hybridization with repetitive DNA probes are reported using a non-enzymatic hybridization technique working with a buffer system not containing any formamide or equivalent chemical denaturing agents. Following simultaneous denaturation of both cells and DNA probes, the renaturation time was reduced to less than 30 min. For one of the DNA probes reasonable FISH-signals were even achieved after about 30 s renaturation time. In addition, the number of washing steps was reduced drastically. As a model system, two repetitive DNA probes (pUC 1.77, D15Z1) were hybridized to human metaphase spreads and interphase nuclei obtained from peripheral blood lymphocytes. The probes were labelled with digoxigenin and detected by FITC-anti-digoxigenin. The hybridization time was reduced step by step and the resulting fluorescence signals were examined systematically. For comparison the pUC 1.77 probe was also hybridized according to a FISH protocol containing 50% formamide. By renaturation for 2 h and overnight two FISH signals per nucleus were obtained. Using shorter renaturation times, no detectable FISH signals were observed. Quantification of the FISH signals was performed using a fluorescence microscope equipped with a cooled colour charge coupled device (CCD) camera. Image analysis was made interactively using a commercially available software package running on a PC (80486). For the pUC 1.77 probe the major binding sites (presumptive chromosomes 1) were clearly distinguished from the minor binding sites by means of the integrated fluorescence intensity. For the two (pUC 1.77) or four (D15Z1) brightest spots on the metaphase spreads and in the interphase nuclei hybridized without formamide, integrated fluorescence intensity distributions were measured for different renaturation times (0.5, 15, 30 min). The intra-nuclear variation in the intensity of the two brightest in situ hybridization spots appeared to be slightly higher (CV between 16 and 32%) than the corresponding variation in the metaphase spreads (CV between 10 and 19%). For the D15Z1 probe FISH signals were detected after hybridization without formamide and 15 min and 30 min renaturation. Always four bright spots were visible and tentatively assigned on the metaphase spreads (presumptive chromosome 15 and 9). The intensity variation of each pair of homologues in a metaphase spread showed a CV of 14 or 15%, respectively, for the presumptive chromosome 15, and 8 or 9%, respectively, for the presumptive chromosome 9.

Analog-Digital Conversion↗
PubMed1994

A rapid FISH technique for quantitative microscopy.

Results of quantitative microscopy for fluorescence in situ hybridization (FISH) signals with repetitive DNA probes (pUC 1.77 and D15Z1) are reported. A nonenzymatic hybridization technique was applied using fluorescein-12-dUTP labeled DNA probes and a buffer system not containing any formamide or equivalent chemical denaturing agents. Following thermal denaturation, the renaturation time was reduced to less than 30 min. The number of wash steps was reduced to one. For the pUC 1.77 probe, the major binding sites (chromosome 1) were distinguished from the minor binding sites by means of fluorescence intensity and spot size. The intensity variation of the two brightest FISH spots (major binding sites) in the same metaphase was 19% for 15 min renaturation time and 16% for 30 min renaturation time. For the D15Z1 probe, generally four bright spots were visible and tentatively assigned according to chromosome length and centromere position (chromosomes 15 and 9). The intensity variation of each two homologues in the same metaphase spread showed a coefficient of variation of 47% (15 min) and 22% (30 min) for chromosome 15, and 19% (15 min) and 15% (30 min) for chromosome 9. The results indicate that the applied technique can considerably accelerate the FISH procedure and is suited for quantitative microscopy.

DNA Probes↗
PubMed1993

Differences of size and shape of active and inactive X-chromosome domains in human amniotic fluid cell nuclei.

It is a widely held belief that the inactive X-chromosome (Xi) in female cell nuclei is strongly condensed as compared to the largely decondensed active X-chromosome (Xa). We have reconsidered this problem and painted X-chromosome domains in nuclei of subconfluent, female and male human amniotic fluid cell cultures (46,XX and 46,XY) by chromosomal in situ suppression (CISS) hybridization with biotinylated human X-chromosome specific library DNA. FITC-conjugated avidin was used for probe detection and nuclei were counterstained with propidium iodide (PI). The shape of these nuclei resembling flat ellipsoids or elliptical cylinders makes them suitable for both two-dimensional (2D) and three-dimensional (3D) analyses. 2D analyses of Xi- and Xa-domains were performed in 34 female cell nuclei by outlining of the painted domains using a camera lucida. Identification of the sex chromatin body in DAPI-stained nuclei prior to CISS-hybridization was confirmed by its colocalization with one of the two painted X-domains. In 31 of the 34 nuclei the area AXi for the inactive X-domain was smaller than the area AXa for the active domain (mean ratio AXa/AXi = 1.9 +/- 0.8 SD, range 1.0-4.3). The signed rank test showed a highly significant (P < .0001) difference both between AXa and AXi and between the ratios r(Xa) and r(Xi), calculated by dividing the maximum length L of each X-domain by its maximum width W. In most nuclei (26/34) we found r(Xa) > r(Xi) demonstrating a generally more elongated structure of Xa. For 3D analysis a confocal scanning laser fluorescence microscope (CSLFM) was used. Ten to 20 light optical sections (PI-image, FITC-image) were registered with equal spacings (approx. 0.4 microns). A thresholding procedure was applied to determine the PI-labeled nuclear and FITC-labeled X-domain areas in each section. Estimated slice volumes were used to compute total nuclear and X-domain volumes. In a series of 35 female nuclei most domains extended from the top to the bottom nuclear sections. The larger of the two X-chromosome domains comprised (3.7 +/- 1.7 S.D.)% of the nuclear volume. A mean ratio of 1.2 +/- 0.2 SD (range 1.1-2.3) was found for the volumes of the larger and the smaller X-domains in these female nuclei. In a series of 27 male amniotic fluid cell nuclei the relative X-chromosome domain volume comprised (4.0 +/- 2.6 S.D.)%.(ABSTRACT TRUNCATED AT 400 WORDS)

Amniotic Fluid↗
PubMed1993

Time-optimized analysis of slit-scan chromosome profiles on a general-purpose personal computer.

Slit-scan flow cytometry provides a method to analyze large numbers of metaphase chromosomes in a relatively short time due to morphological features. The high detection rate requires fast computing for on-line analysis. Up to now, this has been achieved using special-purpose computers, parallel systems or other complex hardware. Here, we describe an algorithm that can be implemented on a general-purpose personal computer. Digitized chromosome profiles can be classified by several criteria especially for the detection of chromosome abnormalities in biological dosimetry. A data set of approximately 4600 profiles was used. Programming in assembler results in an average computing time of about 600 microseconds per profile. Thus on-line evaluation of slit-scanning data appears to become feasible for many flow cytometers running nowadays.

Algorithms↗
PubMed1992

A tilting device for three-dimensional microscopy: application to in situ imaging of interphase cell nuclei.

The resolution of an optical microscope is considerably less in the direction of the optical axis (z) than in the focal plane (x-y plane). This is true of conventional as well as confocal microscopes. For quantitative microscopy, for instance studies of the three-dimensional (3-D) organization of chromosomes in human interphase cell nuclei, the 3-D image must be reconstructed by a point spread function or an optical transfer function with careful consideration of the properties of the imaging system. To alleviate the reconstruction problem, a tilting device was developed so that several data sets of the same cell nucleus under different views could be registered. The 3-D information was obtained from a series of optical sections with a Zeiss transmission light microscope Axiomat using a stage with a computer-controlled stepping motor for movement in the z-axis. The tilting device on the Axiomat stage could turn a cell nucleus through any desired angle and also provide movement in the x-y direction. The technique was applied to 3-D imaging of human lymphocyte cell nuclei, which were labelled by in situ hybridization with the DNA probe pUC 1.77 (mainly specific for chromosome 1). For each nucleus, 3-D data sets were registered at viewing angles of 0 degrees, 90 degrees and 180 degrees; the volumes and positions of the labelled regions (spots) were calculated. The results also confirm that, in principle, any angle of a 2 pi geometry can be fixed for data acquisition with a high reproducibility. This indicates the feasibility of axiotomographical microscopy of cell nuclei.

Cell Nucleus↗
PubMed1992

Automated detection of radiation-induced chromosome aberrations following fluorescence in situ hybridization.

The cytogenetic detection of rare chromosome aberrations induced by ionizing radiation requires the evaluation of large numbers of cells. In this report, an image analysis program based on thresholding of grey level histograms is described for a rapid automated detection of chromosome translocations in metaphase spreads from human lymphocytes following irradiation and chromosome in situ suppression (CISS) fluorescence in situ hybridization (FISH). To classify a metaphase spread as "normal", "aberrant", or "excluded", a minimum medium time of less than two seconds was required on a general purpose Personal Computer. Using appropriately stained specimen, for the false classification rate an upper limit of about 10% was estimated. The upper limit for the rate of "excluded" cells was estimated to be about 11% (99% confidence ranges). FISH-procedures allow to score chromosome aberrations also in the interphase nucleus. To apply simple threshold algorithms for segmentation of the FISH-stained nuclear areas, it is required that the grey level contrast is sufficiently high. The preliminary results presented here on the image analysis of human lymphocyte nuclei suggest the feasibility of such an approach.

Chromosomes↗
PubMed1992

An adapter for defined sample volumes makes it possible to count absolute particle numbers in flow cytometry.

A device is described which makes it possible to count absolute particle (cell) numbers per volume by flow cytometry. It can easily by adapted to several types of flow cytometers, especially to the Coulter EPICS V and EPICS 750 series. A volume adapter has been installed in place of the normal sample handling system without any further modifications of the instrument or the data acquisition program. The adapter consists of a special pipette with two opto-electronic detectors for the beginning and end of the measuring period. These switch on/off a shutter for the illuminating laser beam so that acquisition of the data is controlled indirectly. Sample volumes of 50 microliters were measured at flow rates up to 10(3) particles/s. Calibration beads as well as blood cells were enumerated according to FALS (forward angle light scatter), to SSC (90 degrees light scatter), and to fluorescence parameters. The results were compared to the evaluation made on a Coulter counter or in a Neubauer chamber of a light microscope. Using a concentration of 1 x 10(5)-5 x 10(5) particles/ml, the absolute numbers of particles were determined with a high reproducibility and an estimated error rate of 2-5%.

Antibodies, Monoclonal↗
PubMed1992

PCR amplification and simultaneous digoxigenin incorporation of long DNA probes for fluorescence in situ hybridization.

Nonradioactive in situ hybridization has found widespread applications in cytogenetics. Basic requirements are DNA probes in sufficient amounts and of high specificity as well as a labeling protocol of good reproducibility. The PCR has been of fundamental importance for the amplification of DNA sequences and thus for the production of DNA probes. Meanwhile, PCR protocols for amplification of DNA have reached a high degree of automation. So far, incorporation of labeled nucleotides into these DNA probes has normally been done by nick translation. Here we show that in using the PCR, amplification of a DNA probe larger than one kilobase accompanied by simultaneous incorporation of digoxigenin-11-dUTP can be performed for in situ hybridization experiments. As an example, the DNA probe pUC 1.77 specific for the subcentromeric region q12 of chromosome number 1 was used and hybridized against metaphase chromosomes from human lymphocytes. The labeled chromosome region was detected by anti-digoxigenin-fluorescein, Fab fragments. The experiments were evaluated by digital image analysis of microphotographs.

Chromosomes, Human, Pair 1↗
PubMed1991

Background and peak evaluation of one parameter flow karyotypes on a PC/AT computer.

Flow cytometry has become a fast, quantitative method for the classification of metaphase chromosomes in suspension (flow karyotyping) stained with fluorescent dyes. Such a flow karyotype (frequency distribution of the fluorescence signals) consists of several peaks. The peak pattern characterizes the analyzed chromosome complement. In many cases flow karyotypes contain a continuum of an unspecific background deriving from chromosome fragments or chromosome aggregates. For the quantitative evaluation of a flow karyotype this background has to be subtracted by a suitable background function. In this approach the application of chi 2-functions is described. The feasibility of this method to flow karyotypes has been concluded from a computer simulation of chromosome breaking under different conditions. In spite of the rather rough assumptions of the model compared to the complex reasons that influence chromosome breaking, the chi 2-function fits the background better than the exponential function in current use. The approximation of a Gaussian distribution function by the chi 2-function also makes it possible to use the same subtraction procedure for chromosome aggregates. The procedure was tested for isolated chromosomes of Chinese hamster cell lines under different states of breaking. For further evaluation of one parameter flow karyotypes a setup of computer routines has been developed for PC/AT and compatible computer systems. Different peak values of these flow karyotypes can be determined (e.g. peak mean, standard deviation, absolute and relative peak area etc.). The applied method is to fit Gaussian curves to each peak of an experimentally measured histogram by using an interactive program. Fluctuations depending on 'noise' may be suppressed by a 'k-nearest-neighbours' smoothing procedure.

Animals↗
PubMed1990

Rapid metaphase and interphase detection of radiation-induced chromosome aberrations in human lymphocytes by chromosomal suppression in situ hybridization.

Chromosomal in situ suppression (CISS)-hybridization of biotinylated phage DNA-library inserts from sorted human chromosomes was used to decorate chromosomes 1 and 7 specifically from pter to qter and to detect structural aberrations of these chromosomes in irradiated human peripheral lymphocytes. In addition, probe pUC1.77 was used to mark the 1q12 subregion in normal and aberrant chromosomes 1. Low LET radiation (60Co-gamma-rays; 1.17 and 1.33 MeV) of lymphocyte cultures was performed with various doses (D = 0, 2, 4, 8 Gy) 5 h after stimulation with phytohaemagglutinin. Irradiated cells were cultivated for an additional 67 h before Colcemid arrested metaphase spreads were obtained. Aberrations of the specifically stained chromosomes, such as deletions, dicentrics, and rings, were readily scored after in situ hybridization with either the 1q12 specific probe or DNA-library inserts. By the latter approach, translocations of the specifically stained chromosomes could also be reliably assessed. A linear increase of the percentage of specifically stained aberrant chromosomes was observed when plotted as a function of the square of the dose D. A particular advantage of this new approach is provided by the possibility to delineate numerical and structural chromosome aberrations directly in interphase nuclei. These results indicate that cytogenetic monitoring of ionizing radiation may be considerably facilitated by CISS-hybridization.

Chromosome Aberrations↗
PubMed1990

Real-time multiprocessing of slit scan chromosome profiles.

The multiprocessor NERV and its application to slit scan flow cytometry is described. Up to 320 processors and 640 MBytes of RAM may be used in one VME crate, providing a computing power of less than or equal to 1300 MIPS. The multiprocessor is controlled by a host computer that provides a friendly user interface and comfortable program development tools. All hardware and software has been tested on a prototype NERV system with 5 processors. For a real-time classification/detection of normal and aberrant chromosomes, the centromeric index or the number of centromeres are computed or specifically labeled DNA sequences are detected. The program is partitioned into 60 tasks that can be executed concurrently. A total analysis time of less than 600 microseconds including system overhead will be achieved according to timing measurements which have been done for all individual tasks.

Algorithms↗
PubMed1990

Distribution of chromosome 18 and X centric heterochromatin in the interphase nucleus of cultured human cells.

In situ hybridization of human chromosome 18 and X-specific alphoid DNA-probes was performed in combination with three dimensional (3D) and two dimensional (2D) image analysis to study the interphase distribution of the centric heterochromatin (18c and Xc) of these chromosomes in cultured human cells. 3D analyses of 18c targets using confocal laser scanning microscopy indicated a nonrandom disposition in 73 amniotic fluid cell nuclei. The shape of these nuclei resembled rather flat cylinders or ellipsoids and targets were preferentially arranged in a domain around the nuclear center, but close to or associated with the nuclear envelope. Within this domain, however, positionings of the two targets occurred independently from each other, i.e., the two targets were observed with similar frequencies at the same (upper or lower) side of the nuclear envelope as those on opposite sides. This result strongly argues against any permanent homologous association of 18c. A 2D analytical approach was used for the rapid evaluation of 18c positions in over 4000 interphase nuclei from normal male and female individuals, as well as individuals with trisomy 18 and Bloom's syndrome. In addition to epithelially derived amniotic fluid cells, investigated cell types included in vitro cultivated fibroblastoid cells established from fetal lung tissue and skin-derived fibroblasts. In agreement with the above 3D observations 18c targets were found significantly closer (P less than 0.01) to the center of the 2D nuclear image (CNI) and to each other in all these cultures compared to a random distribution derived from corresponding ellipsoid or cylinder model nuclei. For comparison, a chromosome X-specific alphoid DNA probe was used to investigate the 2D distribution of chromosome X centric heterochromatin in the same cell types. Two dimensional Xc-Xc and Xc-CNI distances fit a random distribution in diploid normal and Bloom's syndrome nuclei, as well as in nuclei with trisomy X. The different distributions of 18c and Xc targets were confirmed by the simultaneous staining of these targets in different colors within individual nuclei using a double in situ hybridization approach.

Amniotic Fluid↗
PubMed1990

Denaturation behaviour of DNA-protein-complexes detected in situ in metaphase chromosomes in suspension by Hoechst 33258 fluorescence.

The denaturation behaviour of DNA-protein complexes in metaphase chromosomes in suspension was analysed in situ by Hoechst 33258 fluorescence. The results indicate that due to the stability of the dye molecule and the product of the molecular extinction coefficient and the quantum yield at different temperatures, Hoechst 33258 is a suitable probe for the detection of double-stranded DNA. Thus, it is possible to monitor the concentration of double-stranded DNA in a suspension by measuring the total fluorescence intensity. The fluorescence denaturation profiles of DNA (calf thymus) were found to be comparable to absorption measurements. The decrease in fluorescence of metaphase chromosomes in suspension with increasing temperature may therefore be used to detect conformational changes of DNA in situ.

Animals↗