Spreading and staining of human metaphase chromosomes on aminoalkylsilane-treated glass slides

Summary The properties of aminoalkylsilane-treated glass slides for the preparation of metaphase spreads and their staining quality have been studied and compared with those of slides which had only been cleaned in ethanol/ether. The parameters investigated were: (1) the average area of metaphases from cultures of blood from both healthy donors and haematology patients; (2) the influence of the positively charged coating on the quality of quinacrine- and Giemsa-banding patterns; (3) non-specific background staining for these banding methods; (4) the number of metaphases as compared to the number of interphase cell nuclei per area of preparation; and (5) the Feulgen-staining intensities of chromosomes and chicken erythrocyte nuclei.The quality of metaphase preparations and the differential staining of chromosomes is better on aminoalkysilane-treated glass slides than that of preparations on routinely cleaned normal microscope slides. In the preparations on aminoalkylsilance-treated slides, the distribution of the cells over the glass surface is more homogeneous; and no influence could be detected on the relative frequency of metaphases as compared to the number of non-divided cell nuclei; the average area per metaphase is increased by about 10% and consequently the number of overlapping chromosomes is decreased.Preparations on aminoalkylsilane-treated glass, after Q-, G- and DAPI-banding procedures, always showed less binding of the staining compounds to the glass slide (a cleaner background) than those on routinely cleaned microscope glass slides. The Feulgen-pararosaniline staining intensities of human metaphase chromosomes and chicken erythrocyte nuclei are the same on aminoalkylsilane-treated slides and on routinely cleaned glass slides. Furthermore, the reproducibility and constancy of quinacrine banding was improved by development of an equilibrium staining method which does not require a washing procedure. The medium, containing 0.002% quinacrine, allows optimal staining results to be obtained for microphotography purposes within 30 min of staining (for visual inspection at least 90 min is required) and is used as the embedding medium.In combination with aminoalkylsilane-treated glass slides, this procedure leads to a clean background and reproducible banding patterns of excellent quality, the results being better and more constant than those of methods described before.     

Induction of diplochromosomes in mammalian cells by inhibitors of topoisomerase II

Diplochromosomes, consisting of four chromatids lying side-by-side, instead of the normal two, are produced when cells go through two rounds of DNA replication without separation of chromatids. They are thus an indication of the failure of the normal chromosome separation mechanism. In the present experiments, induction of diplochromosomes by inhibitors of topoisomerase II (Topo II) was used to provide further evidence that Topo II is required for separation of daughter chromosomes. Actively growing cultures of CHO cells were treated with Colcemid, and separated into metaphase and interphase fractions, each of which was treated for 2 h with the Topo II inhibitor being tested. The cells were then cultivated in fresh medium without inhibitor for periods of between 18 and 44 h, and metaphase cells once again accumulated by treatment with Colcemid. Chromosome preparations were made in the standard way and stained with Giemsa. Up to 2,000 metaphases were counted from each culture, and the proportion with diplochromosomes calculated. At appropriate concentrations, the Topo II inhibitors etoposide and mitoxantrone induced substantial levels of metaphases with diplochromosomes in cultures that had been treated when the cells were in interphase (up to 30% and 11%, respectively). Amsacrine, however, only produced a smaller proportion (4.7%) of metaphases with diplochromosomes after a much longer culture period following treatment. All the inhibitors caused severe chromosome damage. When used to treat metaphase cells, mitoxantrone and amsacrine only induced diplochromosomes after prolonged culture, although a small number of diplochromosomes were seen after etoposide treatment and a shorter period of culture. Results with cells treated in metaphase might indicate that Topo II is, in fact, not required for anaphase chromosome separation, although it is clearly important for segregation of newly replicated DNA. Received: 8 August 1998 / Accepted: 13 September 1998     

Time-saving in biological dosimetry by using the automatic metaphase finder Metafer2.

The amount of time-saving by using the Metafer2 metaphase finder for routine analysis of radiation-induced chromosome aberrations (biological dosimetry) was determined. Metaphases were prepared by standard methods from cultures of human peripheral blood lymphocytes and stained either with Giemsa or with the FPG method. The metaphase finder was used for detecting metaphases on the microscope slides and for automatically processing the evaluation data. In our laboratory, standardized analysis of 1000 metaphases requires at least 3 working days for cell culturing and slide preparation and 51.5 working hours for cytogenetic analysis. When using the metaphase finder the time required for cytogenetic analysis is reduced to 17.3 working hours (time-saving factor: 51.5/17.3 h = 3.0). In our prolonged method, including more than one scoring of each slide and karyotyping of metaphases with chromosome aberrations, the analysis times for 1000 cells are 132 and 70 working hours, respectively (time saving factor: 132/70 h = 1.9).     

Automated metaphase finding: an assessment of the efficiency of the METAFER2 system in a routine mutagenicity assay

The efficiency of the automated metaphase finding system METAFER2 is assessed in a routine mutagenicity assay using an aneuploid rat liver cell line treated with various promutagens. Data sets generated by automated and manual selection of metaphases are compared. It is demonstrated that METAFER2 routinely allows an efficient automatic identification of metaphases not only in lymphocyte preparations, but also in preparation from mammalian cell lines with varying chromosome numbers. Although larger slide areas are required for automated compared to manual metaphase scanning, the automatic system is faster by a factor of about 5. The interactive visual elimination of metaphases of insufficient quality is an easy and fast procedure.METAFER2 allows an unbiased selection of metaphases irrespective of their appearance as homogeneously stained first or harlequin-staines second division cells. Random selection of metaphases is neither influenced by various structural chromosome changes nor by increased frequencies of sister-chromatid exchanges.     

Biological dosimetry by the triage dicentric chromosome assay: potential implications for treatment of acute radiation syndrome in radiological mass casualties

Biological dosimetry is an essential tool for estimating radiation dose. The dicentric chromosome assay (DCA) is currently the tool of choice. Because the assay is labor-intensive and time-consuming, strategies are needed to increase throughput for use in radiation mass casualty incidents. One such strategy is to truncate metaphase spread analysis for triage dose estimates by scoring 50 or fewer metaphases, compared to a routine analysis of 500 to 1000 metaphases, and to increase throughput using a large group of scorers in a biodosimetry network. Previously, the National Institutes for Allergies and Infectious Diseases (NIAID) and the Armed Forces Radiobiology Research Institute (AFRRI) sponsored a double-blinded interlaboratory comparison among five established international cytogenetic biodosimetry laboratories to determine the variability in calibration curves and in dose measurements in unknown, irradiated samples. In the present study, we further analyzed the published data from this previous study to investigate how the number of metaphase spreads influences dose prediction accuracy and how this information could be of value in the triage and management of people at risk for the acute radiation syndrome (ARS). Although, as expected, accuracy decreased with lower numbers of metaphase spreads analyzed, predicted doses by the laboratories were in good agreement and were judged to be adequate to guide diagnosis and treatment of ARS. These results demonstrate that for rapid triage, a network of cytogenetic biodosimetry laboratories can accurately assess doses even with a lower number of scored metaphases.     

The use of harlequin staining to measure delay in the human lymphocyte cell cycle induced by in vitro X-irradiation

Samples of human peripheral blood were given X-ray doses of 1, 2, 3 or 4 Gy at 37°C with a further sample remaining unirradiated. Lymphocytes were then stimulated to divide in cultures containing BrdU for 40–72 h. After harlequin staining the metaphases were recorded as being in their 1st, 2nd or 3rd post-irradiation division. It was confirmed that irradiation delays the proliferation of lymphocytes in culture. A linear relationship between dose and mitotic delay of approximately 1 h per Gray was obtained.This finding of a small effect on cell proliferation is particularly important for biological dosimetry. All in vivo exposures are more or less non-uniform and the lymphocytes in a blood sample therefore possess a spectrum of induced delay characteristics. However, in the great majority of overdose investigations it should not be necessary to increase the normal culture time for the most highly irradiated cells to reach metaphase.The trend towards using harlequin preparations to ensure that only first-division cells are analysed is briefly discussed and it is noted that in this experiment 2nd-cycle metaphases accounted for a maximum of 14% of the cells scored after 48 h in culture.     

The length of the Y chromosome in Nubian males and its location in metaphase spreads

Summary A total of 242 metaphase plates from the peripheral blood of Nubian males living near Aswan, Egypt were studied with respect to the length of the Y chromosome and its location in metaphase spreads. The length of the Y was similar to that found in American Negroes, and the Y chromosome was peripherally located in 79 of the 242 cells.     

T-lymphocytes with 7;14 translocations: frequency of occurrence, breakpoints, and clinical and biological significance.

Among 11,915 consecutive patients and 37 normal controls who had chromosome analysis at the Mayo Clinic between 1978 and 1984, 83 had a single sporadic metaphase with a 7;14 translocation. In 81 of the translocations, the breakpoints were at 14q11 and either 7q34 (type I) or 7p13 (type II): type I translocations occurred in 42 patients, and type II, in 39. The two other translocations had different breakpoints: one was t(7;14)(q11;q32), and the other was t(7;14)(p13;q32). All type I and type II translocations occurred in phytohemagglutinin-stimulated lymphocyte cultures; their combined incidence was 4.88 X 10(-4) per metaphase (81 of 165,991 metaphases) in such cultures. No type I or II translocation was found among 6,713 fibroblast metaphases, 33,463 amniocyte metaphases, or 68,972 bone marrow and unstimulated peripheral blood metaphases. One variant 7;14 translocation occurred in a phytohemagglutinin-stimulated culture, and the other occurred in a fibroblast culture. We did not find a correlation of sporadic 7;14 translocations with any month or season of the year or with patient age or sex. Of the 83 patients, 78 had various clinical disorders, three had ataxia-telangiectasia, one was a normal control, and one was an artificial insemination donor. Follow-up studies on 64 (77%) patients indicate that, to date, none have developed any malignant process subsequent to chromosome analysis. Except for ataxia-telangiectasia, the occurrence of types I and II translocations in lymphocyte cultures may have little, if any, clinical significance. The biological significance of these translocations may be the association of genes in chromosome bands 14q11, 7p13, and 7q34 with the normal physiology of lymphocytes such as the alpha- and beta-chains for T-cell antigen receptor.     

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.     

A Blaze-Dry Spreading Procedure for the Electron Microscopy of Chromosomes from Acid Alcohol-Fixed Human Lymphocytes

A procedure is described for the blaze-drying of human lymphocyte chromosomes on carbonized Parlodion film. Films are prepared by applying Parlodion solution to sheets of freshly cleaved mica. Damage to the film during blaze-drying is prevented by chilling the mica sheets on dry ice before flaming. After spreading, the film and metaphases are floated free from the mica and transferred to a slide of Formvar-coated electron microscope grids. The resulting preparations yield complete metaphase spreads and banded chromosomes morphologically similar to those observed with the light microscope.     

Stepwise accumulation of autoregulated enzyme activities during the cell cycle of the eucaryote Chlorella

Although the allocyclic X chromosome of the mouse frequently appears heteropycnotic, showing chromatid apposition, the feature is not necessarily consistent in conventionally stained metaphases. In contrast, the quinacrine mustard fluorescence and acetic saline Giemsa techniques unambiguously delineate the asynchronously replicating, allocyclic X in most metaphase spreads prepared from 6.5–7.5 days old female embryos, where the allocyclic X is characterized by bright fluorescence over the entire length with two faintly dark bands in the lower middle regions. The same X shows heavy staining by the ASG technique, resulting in less conspicuous banding pattern as compared with its isocyclic homologue. The above features are confirmed in adult bone marrow cells and primary fibroblast culture of the lung derived from the adult female as well as the 18-day-old embryo, though the frequency of cells with an identifiable allocyclic X decreases to some extent. Length measurement demonstrates that the allocyclic X is slightly shorter than the isocyclic one.     

On correcting the error due to metaphase degeneration in stathmokinetic studies

Metaphases arrested by stathmokinetic agents have a limited life span. This may have to be taken into account in drawing conclusions from experiments whose duration is of the same order as the average metaphase life span, or, in the case of a combined arrest and labelling experiment, if G2 is of long duration. The average life span of arrested metaphases in the tissue under study can be estimated by a simple experiment which isolates the G2 cohort. This enables the investigator to decide whether he can trust his metaphase count without correction for loss due to degeneration. If the proposed experiment requires an accumulation of arrested metaphases over a period longer than the average life span of an arrested metaphase there is a serious risk of under-estimating the metaphase index. We propose a simple method of assessing the magnitude of this risk, and making the necessary correction. Assuming that metaphase degeneration is a random process a corrected index of arrested metaphases can be found for expanding populations multiplying the observed metaphase arrest index by exp [(λ-β) (t-m)], where m is the minimum life span of an arrested metaphase, t is the time point from the onset of the experiment, and β and λ are rates at which metaphases are formed and degenerate, respectively. A slight modification of this formula adapts it to steady state systems.     

Establishment of a cell line derived from a canine prostate carcinoma with a highly rearranged karyotype

Akin to the situation in humans, dogs are frequently affected by tumors of the prostate. The malignancies share many similarities between both species, for example, median age at the onset of the disease and metastatic behavior. In human prostatic tumor samples, investigations of prepared metaphase spreads showed a variety of chromosomal aberrations, with trisomies of chromosomes 7, 8, and 17 as the leading cytogenetic abnormalities. In this article we present one case of a canine adenocarcinoma of the prostate, including clinical examination and establishment of a cell line from a tumor sample obtained from the affected 10-year-old male Briard. Searching for similarities between both species in respect to chromosomal changes within the tumor samples, we investigated prepared metaphases of the canine cell line cytogenetically. These investigations presented a highly rearranged karyotype showing a large biarmed marker consisting of material from chromosomes 1 and 2 in addition to centromeric fusions between dog chromosomes 1 and 5 that both could be identified in every metaphase investigated, while centric fusions of chromosomes 4 and 5 occurred in up to 50% of the metaphases. The cell line grew very well and showed evidence of being spontaneously immortalized when it crossed the 20th passage.     

Chromosomal in situ suppression hybridization of human gonosomes and autosomes and its use in clinical cytogenetics

Summary DNA libraries from sorted human gonosomes were used selectively to stain the X and Y chromosomes in normal and aberrant cultured human cells by chromosomal in situ suppression (CISS-) hybridization. The entire X chromosome was stained in metaphase spreads. Interphase chromosome domains of both the active and inactive X were clearly delineated. CISS-hybridization of the Y chromosome resulted in the specific decoration of the euchromatic part (Ypter-q11), whereas the heterochromatic part (Yq12) remained unlabeled. The stained part of the Y chromosome formed a compact domain in interphase nuclei. This approach was applied to amniotic fluid cells containing a ring chromosome of unknown origin (47,XY; +r). The ring chromosome was not stained by library probes from the gonosomes, thereby suggesting its autosomal origin. The sensitivity of CISS-hybridization was demonstrated by the detection of small translocations and fragments in human lymphocyte metaphase spreads after irradiation with ⁶⁰Co-gamma-rays. Lymphocyte cultures from two XX-males were investigated by CISS-hybridization with Y-library probes. In both cases, metaphase spreads demonstrated a translocation of Yp-material to the short arm of an X chromosome. The translocated Y-material could also be demonstrated directly in interphase nuclei. CISS-hybridization of autosomes 7 and 13 was used for prenatal diagnosis in a case with a known balanced translocation t(7;13) in the father. The same translocation was observed in amniotic fluid cells from the fetus. Specific staining of the chromosomes involved in such translocations will be particularly important, in the future, in cases that cannot be solved reliably by conventional chromosome banding alone.Dedicated to Professor Friedrich Vogel on the occasion of his 65th birthday     

A quantitative study of the second meiotic metaphase in male mice (Mus musculus).

Over 11,000 second meiotic metaphase spreads stained for the pericentromeric region have been studied quantitatively in male mice of 14 strains. The sex-chromosome constitution of a cell could be judged objectively if X and Y chromosomes and ploidy were all scored. A bias arose if only Y chromosomes and ploidy were scored but could be corrected statistically. There was no sign of other forms of bias. The original contiguity of X and Y second metaphases in vivo was very occasionally evident in the preparations. Most of the subhaploid aneuploid counts were assumed to be artifactual. The incidence of truly aneuploid second metaphases in 13 strains was estimated as 0.38+/-0.12%. The estimated average rate per chromosome was 0.019+/-0.006%, with a comparable order of magnitude for the sex chromosomes alone. Simultaneous aneuploidy of two or more chromosomes of the haploid set was estimated to be very rare. Of the spreads from 13 strains, 9.6% were polyploid (2N, 3N, 4N) and showed most of the possible combinations of sex chromosomes. Nearly all the polyploid spreads were considered to arise by artifactual cell fusion at the time of second metaphase during the preparative technique, especially of the X and Y daughter-cell products of the first meiotic division. Other modes of origin (true polyploidy, accidental superposition of cells during preparation) were unlikely. The data could be accommodated by a statistical model with only four parameters. It allowed for artifactual fusion mainly between daughter cells but also between non-daughter cells, bias in one scoring method, and bias in the numbers of cells with given ploidy successfully mounted. Current techniques of chromosome preparation were thought to be wholly unsuitable for the recognition of true polyploidy. The artifactual origin of polyploid spreads was borne out by an absence of polyploid spermatozoa in 14 strains. There appeared to be a virtually constant transmission rate of paternal X and Y chromosomes from early meiosis to late blastocyst. The estimated rate of 49.05+/-0.67% with a Y chromosome also estimated the primary sex ratio. There was evidence of polymorphism in autosomal pericentromeric staining in 3 strains. No measure of the numbers of autosomes or sex chromosomes varied significantly between duplicate preparations or between duplicate males of a strain.     

5-Bromodeoxycytidine in the study of sister chromatid exchanges in human lymphocytes

Summary When [³H]dC was added with a high dose (4x10^-1 mM) of dT to human blood lymphocyte cultures, much heavier labeling of interphase nuclei and metaphase chromosomes was observed compared with that in cultures treated with [³H]dC alone. This observation indicates that in the presence of excess dT, exogenous dC is included into cytosine bases of DNA, releasing the cells from the thymidine block.BrdC 5x10^-2 mM added with a high dose of dT (4x10^-1 to 1.0 mM) to the cultures did not relieve the thymidine block as determined from the percentage of metaphases of the first to third divisions. It is concluded that BrdC, in contrast to dC, is not utilized as a cytosine DNA precursor even in the presence of high concentrations of dT.The frequency of SCEs per cell was the same when studied with the aid of BrdC and BrdU used under similar conditions. The distribution of SCEs among chromosomes was also identical for both analogues: The number of SCEs was significantly higher than expected in chromosomes of group B and lower than expected in chromosomes of groups E, F, and G.     

Chromosome aberrations induced in human lymphocytes by in vitro and in vivo X-rays

A dose-effect curve is presented obtained by analysis of dicentric chromosomes and centric ring chromosomes in lymphocyte metaphase spreads of three healthy volunteers after in vitro 100 kV X-ray-irradiation of peripheral blood samples. This calibration curve follows a linear quadratic equation, y=c+alpha D+beta D(2), with the coefficients: y=(0.0005+/-0.0001)+(0.0355+/-0.0066)D+(0.0701+/-0.0072)D(2). The model is based on 13.231 first-division metaphases analyzed after in vitro exposure to doses ranging from 0.1 to 2.0 Gy at a dose rate of 0.4 Gy min(-1). Significant overdispersion of the observed chromosomal aberrations was evident for dose points 1.0 and 2.0 Gy, respectively. The calibration curve was applied to derive equivalent whole body doses of three subjects after suspected extensive exposure to diagnostic X-rays.     

High-resolution chromosomes of rhesus macaques (Macaca mulatta)

Late-prophase high-resolution chromosomes were successfully cultured for 22 of 27 Macaca mulatta samples. Twelve of the successful cultures were adequate for karyotyping high-resolution spreads. High-resolution chromosome technique provides an important contribution to primate genetics because it can be used to identify chromosomal anomalies undetected in metaphase spreads and may be useful for paternity exclusion analysis.     

A comparison of aberration distribution and cell-cycle progression in cells treated with bleomycin with those exposed to X-rays

The extent of cell-cycle delay and the frequency of aberrant metaphases induced by bleomycin (BLM) and X-rays have been compared at doses which produce similar frequencies of chromosome aberrations by the 2 clastogenic agents (BLM, 40 micrograms/ml and X-rays, 2 Gy) in muntjac lymphocytes. The frequency of aberrant metaphases was low in BLM-treated cells; however, the number of aberrations per metaphase was higher than in cells exposed to X-rays. Thus in contrast to their uniform sensitivity to X-rays, the lymphocytes showed differential sensitivity to BLM. This might be due to differences among the cells in their uptake of BLM and/or its action on the nuclear membrane-DNA complex. In spite of the total number of chromosome aberrations being similar to that induced by X-rays, BLM did not induce a significant delay in cell-cycle progression as observed in the case of X-rays. A possible explanation could be that the DNA damages being limited to fewer cells than in the case of X-irradiation, the BLM-treated cultures had more normal cells allowing faster progression and/or unlike X-rays BLM may not be causing other cellular damages in addition to DNA breaks.     

Cytogenetic abnormalities in a patient with hypercalcemia and papillary thyroid carcinoma

Summary Cytogenetic examinations on multiple peripheral blood cultures of a patient with papillary thyroid carcinoma and hypercalcemia revealed the following features: (1) The average frequency of cells with aberrations was 11.6%, considerably higher than in controls. Among metaphases with chromosomal abnormalities, 4.5% had chromosome-type aberrations. (2) One homolog of chromosome 11 showed a fragile site in the proximal end of the long arm, and in three metaphases the segment distal to the fragile site showed branched morphology. (3) The rate of sister chromatid exchanges was within normal limits (8.78/metaphase). (4) The patient's two sons showed 7.0% and 5.0% abnormal metaphases, in the high normal range.