Chromosome heteromorphism quantified by high-resolution bivariate flow karyotyping.

Maternal and paternal homologues of many chromosome types can be differentiated on the basis of their peak position in Hoechst 33258 versus chromomycin A3 bivariate flow karyotypes. We demonstrate here the magnitude of DNA content differences among normal chromosomes of the same type. Significant peak-position differences between homologues were observed for an average of four chromosome types in each of the karyotypes of 98 different individuals. The frequency of individuals with differences in homologue peak positions varied among chromosome types: e.g., chromosome 15, 61%; chromosome 3, 4%. Flow karyotypes of 33 unrelated individuals were compared to determine the range of peak position among normal chromosomes. Chromosomes Y, 21, 22, 15, 16, 13, 14, and 19 were most heteromorphic, and chromosomes 2-8 and X were least heteromorphic. The largest chromosome 21 was 45% larger than the smallest 21 chromosome observed. The base composition of the variable regions differed among chromosome types. DNA contents of chromosome variants determined from flow karyotypes were closely correlated to measurements of DNA content made of gallocyanin chrome alum-stained metaphase chromosomes on slides. Fluorescence in situ hybridization with chromosome-specific repetitive sequences indicated that variability in their copy number is partly responsible for peak-position variability in some chromosomes. Heteromorphic chromosomes are identified for which parental flow karyotype information will be essential if de novo rearrangements resulting in small DNA content changes are to be detected with flow karyotyping.     

Detection of aneuploidy involving chromosomes 13, 18, or 21, by fluorescence in situ hybridization (FISH) to interphase and metaphase amniocytes.

Fluorescence in situ hybridization (FISH) with chromosome-specific probes has been applied to detection of numerical aberrations involving chromosomes 13, 18, and 21 in metaphase and interphase amniocytes. High-complexity, composite probes for chromosomes 13, 18, and 21 were used as hybridization probes for this study. These probes were constructed as chromosome-specific libraries in Bluescribe plasmids and are designated pBS-13, pBS-18, and pBS-21. Elements of these probes bind at numerous sites along the target chromosome and, when detected fluorescently, stain essentially the entire long arm of the target chromosome. The target chromosome number (i.e., the number of chromosomes of the type for which the probe was specific) was correctly determined in 20 of 20 samples in which metaphase spreads were analyzed and in 43 of 43 samples in which interphase nuclei were analyzed; all of these studies were conducted in blind fashion. These results suggest the utility of FISH with composite probes for rapid detection of numerical aberrations in metaphase and interphase amniotic cells.     

X-ray- and neutron-induced chromosome damage detected by flow cytometry compared to cell lethality and chromosome structural changes

V79 Chinese hamster cells were irradiated in G0 phase with 200 kV X rays or 14 MeV neutrons, and dose-response curves were determined for three end points: chromosome damage detected by flow cytometric analysis of chromosomes isolated from metaphase cells in irradiated cultures; loss of clonogenic capacity; and induction of dicentric, tricentric, and ring chromosomes. The changes observed in the flow karyotypes from irradiated cultures were quantitatively evaluated by computer analysis. Estimates of the frequencies of chromosome lesions were derived from an analysis of the flow cytometric measurements by means of a comparison with model calculations simulating the effect of chromosome changes on flow karyotypes. The results indicate that lesions assayed by flow cytometry occur three times more frequently than lethal lesions, while the chromosomal structural changes detected by microscopic analysis were about 10 times less frequent than the lesions detected by flow cytometry. Dose-response curves for X rays and neutrons show that cell reproductive death and changes in flow karyotypes result from damage, induced with a similar relative biological effectiveness. Dose-effect relations derived from changes in flow karyotypes, which can be obtained within 24 h after irradiation, might be of value as a predictive test for the sensitivity of cells for loss of clonogenic capacity.     

Semi-automated detection of aberrant chromosomes in bivariate flow karyotypes.

A method is described that is designed to compare, in a standardized procedure, bivariate flow karyotypes of Hoechst 33258 (HO)/Chromomycin A3 (CA) stained human chromosomes from cells with aberrations with a reference flow karyotype of normal chromosomes. In addition to uniform normalization of normal and abnormal flow karyotypes, the main purpose is detection of structurally abnormal chromosomes in often complex karyotypes of tumor cells. The method, which has been implemented in a computer program, consists of a comparison of individual chromosome peaks with the positions of peaks in the flow karyotype constituted by normal chromosomes and takes into account the natural variability in base composition of normal chromosomes among healthy individuals. Flow-karyotypes are normalized using an iterative fitting procedure, using corrections for (1) amplification of HO and CA fluorescence, (2) cross-talk between the fluorescence signals of HO and CA, and (3) offset of the HO and CA origin. Flow karyotypes of two cell lines, one with a simple deletion and the other with more complex karyotypic changes, were analyzed. The results of flow analysis were found to be in general agreement with the cytogenetic analysis of quinacrine banded karyotypes.     

Optimising human chromosome separation for the production of chromosome-specific DNA libraries by flow sorting

Summary A number of cell lines, some containing chromosomes with distinctive heteromorphisms, have been flow karyotyped using a single laser flow sorter in an attempt to select those suitable for sorting all human chromosomes individually. Using the non-base-specific DNA stain ethidium bromide, chromosomes 3,4,5, and 6 form individual peaks in practically all normal subjects, while the right combination of heteromorphisms enables chromosomes 1, 2, 8, 9, 13, 16, 17, 18, 19, 20, 21, 22, and Y to be sorted separately. Two male cell lines, one containing a duplication and one a deletion of the X, produce flow karyotypes suitable for sorting chromosomes 7 and 8. The use of numerical chromosome abnormalities to enrich the sex chromosomes and the autosomes 18 and 21 is also illustrated. The DNA stain Hoechst 33258 binds preferentially to AT base pairs. Flow karyotypes produced with this fluorochrome separate some chromosomes not well separated with ethidium bromide. Chromosomes 5, 6, 8, 13, 14, 15, 17, and 20, and Y can be sorted individually with Hoechst 33258 with the right combination of heteromorphisms. Using these techniques, all human chromosomes apart from 10, 11, and 12 have been found as individual flow karyotype peaks, suitable for sorting with a high degree of purity.     

Report on the Eleventh International Workshop on the Identification of Transcribed Sequences 2001. November 9-11, 2001. Washington, DC, USA

Glioblastoma multiforme (GBM) is characterized by intratumoral heterogeneity as to both histomorphology and genetic changes, displaying a wide variety of numerical chromosome aberrations the most common of which are monosomy 10 and trisomy 7. Moreover, GBM in vitro are known to have variable karyotypes within a given tumor cell culture leading to rapid karyotype evolution through a high incidence of secondary numerical chromosome aberrations. The aim of our study was to investigate to what extent this mitotic instability of glioblastoma cells is also present in vivo. We assessed the spatial distribution patterns of numerical chromosome aberrations in vivo in a series of 24 GBM using two-color in situ hybridization for chromosomes 7/10, 8/17, and 12/18 on consecutive 6-microm paraffin-embedded tissue slides. The chromosome aberration patterns were compared with the histomorphology of the investigated tumor assessed from a consecutive HE-stained section, and with the in vitro karyotype of cell cultures established from the tumors. All investigated chromosomes showed mitotic instability, i.e., numerical aberrations within significant amounts of tumor cells in a scattered distribution through the tumor tissue. As to chromosomes 10 and 17, only monosomy occurred, as to chromosome 7 only trisomy/polysomy, apparently as a result of selection in favor of the respective aberration. Conversely, chromosomes 8, 12, and 18 displayed scattered patterns of monosomy as well as trisomy within a given tumor reflecting a high mitotic error rate without selective effects. The karyotypes of the tumor cell cultures showed less variability of numerical aberrations apparently due to clonal adaptation to in vitro conditions. We conclude that glioblastoma cells in vivo are characterized by an extensive tendency to mitotic errors. The resulting clonal diversity of chromosomally aberrant cells may be an important biological constituent of the well-known ability of glioblastomas to preserve viable tumor cell clones under adaptive stress in vivo, in clinical terms to rapidly recur after antitumoral therapy including radio- or chemotherapy.     

Quantification of the DNA content of structurally abnormal X chromosomes and X chromosome aneuploidy using high resolution bivariate flow karyotyping

Quantification of the Hoechst and chromomycin A3 fluorescence intensities of mitotic human chromosomes isolated from karyotypically normal and abnormal cells was performed with a dual beam flow cytometer. The resultant flow karyotypes contain information about the relative DNA content and base composition of chromosomes and their relative frequencies in the mitotic cell sample. The relative copy number of X and Y chromosomes was determined for 38 normal males and females and 6 cell lines with X or Y chromosome aneuploidy. Flow karyotype diagnoses corresponded with conventional cytogenetic results in all cases. We show that chromosome DNA content can be derived from peak position in Hoechst vs. chromomycin flow karyotypes. These values are linearly related to propidium iodide staining intensity as measured with flow cytometry and to the binding of gallocyanin chrome alum to phosphate groups as measured with slide-based scanning photometry. Cell lines with deleted or dicentric X chromosomes ranging in length from 0.53 to 1.95 times normal were analyzed by using flow cytometry. The measured difference in DNA content between a normal X and each of the structurally abnormal chromosomes was linearly correlated to the difference predicted from cytogenetics and/or probe analyses. Deletions of 3-5 Mb, which were at and below the detection limits of conventional cytogenetics, could be quantified by flow karyotyping in individuals with X-linked diseases such as Duchenne muscular dystrophy, choroideremia, and ocular albinism/ichthyosis. The results show that the use of flow karyotyping to quantify the size of restricted regions of the genome can complement conventional cytogenetics and other physical mapping techniques in the study of genetic disorders.     

Algorithms for the evaluation of radiation induced chromosome aberration yields per cell from flow karyotypes

CHO-K1 cells were irradiated in G0/G1-phase with 150 kV X-rays. Single chromosomes isolated from metaphase cells and stained with DNA intercalating dye DAPI were analyzed in the ICP 22 with a modified flow chamber. In order to study dose-dependent changes in the flow karyotypes, they were split into peak- and background-portions by an iterative fit algorithm. As in a first approach, estimates of the frequencies of chromosome lesions were derived from an evaluation of the dose-dependent reduction in peak contents. The number of radiation-induced lesions per chromosome was found to be proportional to its length. As a second approach, the number of fluorescence events in the histogram background was corrected for non-chromosomal debris and evaluated interms of chromosome aberration frequency per cell, which was consistent with the yields of dicentric chromosomes and acentric fragments observed in microscopic investigations. As a third approach, lesion frequencies were calculated from the corrected background light sum in the karyotypes, utilizing a Monte Carlo model to simulate the effect of aberration formation on the flow histogram. The results indicate that the number of chromosome lesions observed by flow cytometry can be quantitatively related to the yield of structural chromosome aberrations detected by microscopic analysis. Dose-effect relations and split-dose kinetics are given as examples demonstrating the usefulness of this technique in radiobiology. Time saving compared to microscopic analysis was of the order of 90%.     

Banded karyotypes of H-4-IIE-C3 rat hepatoma cells grown in vitro.

Mitotic cells from the H-4-IIE-C3 rat hepatoma tissue culture line showed a range of 45 to 53 chromosomes per cell with 75% of the cells displaying a chromosome mumber between 49 and 52. Analysis of Wright's-Giemsa banded karyotypes of 22 cells revealed considerable cell to cell variation. Twenty-one structurally abnormal chromosomes were identified in these cells; the origin of nine of the 21 chromosomes were identified in these cells; the origin of nine of the 21 chromosomes could be determined. Of the structurally abnormal chromosomes detected, only one (M-1) occurred with a sufficiently high frequency to be of general use as a marker for these cells. This marker appears to be a Robertsonian translocation involving chromosome number 2 and chromosome number 10.     

Three major cytogenetic subgroups can be identified among chromosomally abnormal solitary lipomas

Summary We have investigated cytogenetically a total of 35 solitary lipomas, 10 of which have been reported previously. Of the 25 tumours presented herein for the first time, clonal chromosome aberrations were detected in 17. The remaining eight had normal karyotypes, although two of them had nonclonal aberrations in about one quarter of the cells. Based on the cytogenetic findings in all 35 lipomas, four major subgroups can be distinguished. These are characterized by: (I) hyperdiploid karyotypes including one or more supernumerary ring chromosomes (5 cases); (II) diploid karyotypes with mostly balanced rearrangements involving 12q13-14 (13 cases), including the rearrangement t(3;12) (q27-28;q13-14) in 4 cases; (III) hypodiploid or diploid karyotypes with other aberrations than ring chromosomes or rearrangements of 12q13-14 (8 cases); and (IV) normal karyotypes (9 cases).     

Banded karyotypes of H-4-IIE-C3 rat hepatoma cells grown in vitro

Summary Mitotic cells from the H-4-IIE-C3 rat hepatoma tissue culture line showed a range of 45 to 53 chromosomes per cell with 75% of the cells displaying a chromosome number between 49 and 52. Analysis of Wright’s-Giemsa banded karyotypes of 22 cells revealed considerable cell to cell variation. Twenty-one structurally abnormal chromosomes were identified in these cells; the origin of nine of the 21 chromosomes were identified in these cells; the origin of nine of the 21 chromosomes could be determined. Of the structurally abnormal chromosomes detected, only one (M-1) occurred with a sufficiently high frequency to be of general use as a marker for these cells. This marker appears to be a Robertsonian translocation involving chromosome number 2 and chromosome number 10. This work was supported by grants-in-aid made available through the San Diego State University Foundation.     

Reverse chromosome painting for the identification of marker chromosomes and complex translocations in leukemia.

BACKGROUND: Chromosome banding techniques and in situ hybridization reveal the majority of chromosomal aberrations. However, difficulties remain in cases of highly contracted chromosomes, poor quality of the metaphases or the presence of markers with the involvement of several chromosomes. Here, it is demonstrated that reverse painting can be applied successfully starting with bone marrow cells from primary acute myelocytic leukemias (AML). METHODS: This was accomplished by culturing the leukemic cells with a cocktail of various growth factors, which yielded sufficient numbers of cells in cycle to harvest chromosomes for sorting. Aberrant chromosomes were flow-sorted and amplified by degenerate oligonucleotide-primed PCR. The resulting products were labeled by nick-translation and hybridized on normal metaphase spreads. RESULTS: Two patients with marker chromosomes in their leukemia cells were analyzed in detail. The hybridization pattern displayed the composition of the aberrant sorted chromosome. Results were compared with conventional cytogenetic analyses that were performed on material obtained from the same aspirate. The reverse-painting technique enabled identification of aberrations that were not detected by conventional cytogenetic analysis. CONCLUSIONS: Primary AML cells can be cultured in vitro, using optimal culture conditions, facilitating the production of high quality flow karyotypes, suitable for sorting of marker chromosomes to produce DOP-PCR derived chromosome painting probes for reverse painting. Valuable additional cytogenetic information can thus be obtained about complex chromosomal rearrangements or structural aberrations that could not be completely resolved by conventional cytogenetic analysis.     

Analysis and sorting of rye (Secale cereale L.) chromosomes using flow cytometry.

Procedures for chromosome analysis and sorting using flow cytometry (flow cytogenetics) were developed for rye (Secale cereale L.). Suspensions of intact chromosomes were prepared by mechanical homogenization of synchronized root tips after mild fixation with formaldehyde. Histograms of relative fluorescence intensity obtained after the analysis of DAPI-stained chromosomes (flow karyotypes) were characterized and the chromosome content of the DNA peaks was determined. Chromosome 1R could be discriminated on a flow karyotype of S. cereale 'Imperial'. The remaining rye chromosomes (2R-7R) could be discriminated and sorted from individual wheat-rye addition lines. The analysis of lines with reconstructed karyotypes demonstrated a possibility of sorting translocation chromosomes. Supernumerary B chromosomes could be sorted from an experimental rye population and from S. cereale 'Adams'. Flow-sorted chromosomes were identified by fluorescence in situ hybridization (FISH) with probes for various DNA repeats. Large numbers of chromosomes of a single type sorted onto microscopic slides facilitated detection of rarely occurring chromosome variants by FISH with specific probes. PCR with chromosome-specific primers confirmed the identity of sorted fractions and indicated suitability of sorted chromosomes for physical mapping. The possibility to sort large numbers of chromosomes opens a way for the construction of large-insert chromosome-specific DNA libraries in rye.     

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.     

Inheritance of chromosome heteromorphisms analyzed by high-resolution bivariate flow karyotyping.

The DNA content of the mitotic chromosomes from 10 children and their parents in four families were quantified by bivariate flow karyotyping. In all cases, each chromosome peak in the flow karyotype of the child could be traced to one of the two parents. The measured absolute difference in homologue DNA content between children and their parents averaged 0.8%, or approximately 1 Mbp over all chromosome types. This study demonstrates that flow karyotypes of a proband's parents can be an aid in detecting and quantifying the size of de novo deletions that involve heteromorphic chromosome types.     

Variability of chromosomal DNA contents in maize (Zea mays L.) inbred and hybrid lines

The flow karyotypes of different maize (Zea mays L.) inbred and hybrid lines were analyzed. The accumulation and isolation of large quantities of high-quality metaphase chromosomes from root tips was achieved from many kinds of maize lines. The chromosome suspensions were prepared by a simple slicing method from synchronized maize root tips and analyzed by flow cytometry. Variations of experimental flow karyotypes were detected among inbred and hybrid lines in terms of the positions and/or the numbers of chromosome peaks. The 2C DNA amount among eight inbred lines ranged from 5.09 to 5.52 pg. The selection of appropriate maize lines is critical for sorting specific single chromosome types. At least five different chromosome types can be discriminated and sorted from five maize lines. The variability of DNA content in maize chromosome 1 was 9.1%, ranging from 0.685 to 0.747 pg. Differences were detected in the DNA content of homologous chromosome 1 of hybrid lines.     

Preparation and bivariate analysis of suspensions of human chromosomes

Chromosomes were isolated from a variety of human cell types using a HEPES-buffered hypotonic solution (pH 8.0) containing KCl, MgSO4, dithioerythritol, and RNase. The chromosomes isolated by this procedure could be stained with a variety of fluorescent stains including propidium iodide, chromomycin A3, and Hoechst 33258. Addition of sodium citrate to the stained chromosomes was found to improve the total fluorescence resolution. High-quality bivariate Hoechst vs. chromomycin fluorescence distributions were obtained for chromosomes isolated from a human fibroblast cell strain, a human colon carcinoma cell line, and human peripheral blood lymphocyte cultures. Good flow karyotypes were also obtained from primary amniotic cell cultures. The Hoechst vs. chromomycin flow karyotypes of a given cell line, made at different times and at dye concentrations varying over fourfold ranges, show little variation in the relative peak positions of the chromosomes. The size of the DNA in chromosomes isolated using this procedure ranges from 20 to over 50 kilobases. The described isolation procedure is simple, it yields high-quality flow karyotypes, and it can be used to prepare chromosomes from clinical samples.     

Flow karyotyping of human melanoma cell lines

Single-laser flow cytometry has been used to study the feasibility of flow karyotyping of human solid tumors. As a model, seven human melanoma cell lines have been used with varying numerical chromosome composition as verified by FCM DNA content measurements and chromosome countings. For all seven cell lines, flow karyotypes that showed a variety of consistent deviations from the normal diploid flow karyotype could be obtained although the resolution of the flow system and varying debris continuum limited the number of resolvable peaks. The predominant changes observed involved the regions normally representing chromosomes 3-8, 9-12, and 13-16. It is concluded that at present the preparation procedure is the main limiting factor for exploring the full potential of flow karyotyping for cytogenetic analysis of solid-tumor cell lines.     

Determination of the DNA content of human chromosomes by flow cytometry

The mean relative DNA content of each human chromosome was calculated from flow karyotypes of ethidium bromide-stained chromosomes obtained from healthy, normal individuals. These values were found to correlate closely with previously published data obtained by photometric scanning of stained, fixed chromosomes. Calculations of the normal variation in DNA content of each human chromosome indicated that chromosomes 1, 9, 16, and Y (chromosomes with large centric heterochromatic regions) were the most variable, followed by the acrocentrics, 13, 14, 15, 21, and 22. Chromosomes 2, 3, 18, and 19 were also found to vary significantly in DNA content. Chromosomes from a number of subjects with extreme heteromorphisms were flow karyotyped to obtain an estimate of the extent of variation in DNA content of each chromosome. The greatest difference between extreme variants was found for chromosome 1 (which differed by 0.82% of the total genomic DNA), followed by 16 and 9. The largest Y-chromosome variant was 85.9% bigger than the smallest. The precise karyotype analysis produced by flow cytometry resolved many differences between chromosome homologs, including some that cannot be readily distinguished cytogenetically. The implications of these findings for detection of chromosome abnormalities by flow karyotype analysis are discussed.     

Flow sorting of mitotic chromosomes in common wheat (Triticum aestivum L.)

The aim of this study was to develop an improved procedure for preparation of chromosome suspensions, and to evaluate the potential of flow cytometry for chromosome sorting in wheat. Suspensions of intact chromosomes were prepared by mechanical homogenization of synchronized root tips after mild fixation with formaldehyde. Histograms of relative fluorescence intensity (flow karyotypes) obtained after the analysis of DAPI-stained chromosomes were characterized and the chromosome content of all peaks on wheat flow karyotype was determined for the first time. Only chromosome 3B could be discriminated on flow karyotypes of wheat lines with standard karyotype. Remaining chromosomes formed three composite peaks and could be sorted only as groups. Chromosome 3B could be sorted at purity >95% as determined by microscopic evaluation of sorted fractions that were labeled using C-PRINS with primers for GAA microsatellites and for Afa repeats, respectively. Chromosome 5BL/7BL could be sorted in two wheat cultivars at similar purity, indicating a potential of various wheat stocks for sorting of other chromosome types. PCR with chromosome-specific primers confirmed the identity of sorted fractions and suitability of flow-sorted chromosomes for physical mapping and for construction of small-insert DNA libraries. Sorted chromosomes were also found suitable for the preparation of high-molecular-weight DNA. On the basis of these results, it seems realistic to propose construction of large-insert chromosome-specific DNA libraries in wheat. The availability of such libraries would greatly simplify the analysis of the complex wheat genome.