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PubMed1999

Molecular cytogenetic detection of trisomy 21 in interphase nuclei and metaphase chromosomes.

OBJECTIVE: To explore the potential application of molecular methodology in the detection of numerical chromosome aberration for clinical diagnosis and prenatal diagnosis of Down's syndrome. METHODS: The primed in situ labelling (PRINS) procedure was carried out by in situ annealing of specific oligonucleotide primers to chromosome 13, 16, 18, 21, X and Y, respectively, followed by primer elongation by a Taq polymerase in the presence of labelled nucleotides. Detection of the labelling sites was performed by immunocytochemistry and conventional fluorescence microscopy. RESULTS: Under the stringent annealing temperature, chromosomes 13, 16, 18, X and Y were specifically labelled at centromeres and the procedure was carried out successfully on interphase nuclei as well as on metaphase spreads with easily scorable signals. In 33 cases of uncultured peripheral blood lymphocytes and 11 cases of uncultured amniocytes tested, two fluorescence signals were shown on more than 87.6% interphase nuclei when chromosomes 13, 16, 18 were investigated. Sex chromosomes were correctly detected in the same way. Blind tests on peripheral blood lymphocytes from 14 cases of normal individuals and 12 cases of Down's syndrome patients and on 9 cases of amniocytes showed that when chromosome 21 was detected by PRINS, two fluorescence spots as positive signals were visible on 89.3% normal nuclei and three spots on 88.8% trisomic nuclei. The above results were fully compatible with karyotypic analysis. CONCLUSION: PRINS provides a rapid and efficient method for the clinical diagnosis or prenatal diagnosis of trisomy 21.

Amnion↗
PubMed2001

[Detection of BCL2 translocation in an interphase neucleus using fluorescence in situ hybridization strategy].

OBJECTIVE: To establish a specific method to diagnose non-Hodgkin's lymphoma in clinic. METHODS: Fluorescence in situ hybridization (FISH) strategy capable of detecting t(14;18)(q32;q21) chromosome translocation in an interphase nucleus was developed using a YAC clone containing the BCL2 gene and a phage clone containing IgHC(mu) region as probes. In the SU-DHL-6 cells, the translocated BCL2 allele (red) is overlapped by one of the green signals from IgH phage probe, while in normal lymphocytes the least distance of the hybridization signals from two different probes was either equal or larger than one-tenth of the diameter of a nucleus. RESULTS: Based on the relative position of the signals from BCL2 and IgH probes the BCL2 translocation can be clearly detected in an interphase nucleus. The metaphase number and quality in samples, which could significantly restrict the reliability of cytogenetic analysis, can be ignored here. CONCLUSION: This method shows a potentiality in clinical diagnosis of non-Hodgkin's lymphoma because of its objectivity, reliability and simplicity.

Cell Nucleus↗
PubMed2001

Validation of a multicolor interphase fluorescence in situ hybridization assay for detection of transitional cell carcinoma on fresh and archival thin-layer, liquid-based cytology slides.

OBJECTIVE: To evaluate the feasibility of performing multicolor interphase fluorescence in situ hybridization (FISH) on ThinPrep slides of transitional cell carcinoma (TCC). STUDY DESIGN: Slides from 20 voided urine specimens were prepared by the ThinPrep technique (Cytyc, Boxborough, Massachusetts, U.S.A.), pretreated using a pretreatment kit and subjected to hybridization with the multicolor FISH probe UroVysion (Vysis, Downers Grove, Illinois, U.S.A.). Archival slides were placed in xylene, destained in alcohol and washed prior to pretreatment. Urines from patients with cytology-positive, biopsy-proven grade 1 (n = 5), 2 (n = 7) and 3 (n = 5) TCC and negative cytology and biopsy (n = 3) were selected. Freshly prepared (n = 10) and archival (n = 10) slides were used. RESULTS: All carcinoma cases were FISH positive (> 5 cells with complex abnormalities of > or = 2 studied chromosomes per slide). None of the normal samples were aneusomic. Gain of chromosomes 3, 7 and 17 constituted the majority of positive cases. Proper destaining and slight decrease in stringency wash conditions enabled reliable detection of signals in archival cases. CONCLUSION: Routine ThinPrep slides can be used for multicolor interphase FISH analysis of urine cytology specimens. Archival slides provide the opportunity to analyze by FISH the nature of atypical cells identified by cytology. This revised method allows FISH technology more accessibility for routine use in cytology laboratories.

Carcinoma, Transitional Cell↗
PubMed2001

Interphase FISH--assisted second-trimester termination of a trisomy 21 fetus in an IVF-ET twin pregnancy. A case report.

BACKGROUND: When confronting a dizygotic pregnancy with one fetus affected with chromosomal aberrations, most couples would opt for selective termination of the affected twin. CASE: Routine genetic amniocentesis was performed for an in vitro fertilization-embryo transfer twin pregnancy at 18 weeks' gestation due to advanced maternal age. After two weeks, cytogenetic analysis revealed that both twins were male and one was affected with trisomy 21. At that time, ultrasound examination could not tell them apart with certainty. With the aid of interphase fluorescence in situ hybridization (FISH), we had no trouble locating the affected twin and performed feticide successfully with an intracardiac potassium chloride injection. At 37 weeks of gestation, a normal male was delivered along with a macerated trisomy 21 fetus. CONCLUSION: In a dizygotic twin pregnancy discordant for chromosome aberrations, when ultrasound cannot distinguish the affected twin, performing interphase FISH with an appropriate chromosome probe proves very useful in quickly and accurately locating the chromosomally abnormal twin for selective termination.

Adult↗
PubMed2002

Evidence for interphase DNA decondensation transverse to the chromosome axis: a multicolor banding analysis.

In the present study a multicolor banding (MCB) analysis was performed to address the up to now unrequited question in which direction with respect to the axis chromosomes decondense in interphase. It could be demonstrated, that i) MCB produces a similar banding pattern in interphase as in metaphase; ii) that no complete decondensation and dispersion appears, which is in concordance with the concept of chromosome territories; and iii) chromosome decondensation happens square to chromosome axis. The presented data are important for a better understanding of nuclear architecture, however, further studies are required.

Chromosome Banding↗
PubMed2002

[Detection of inv (16) in acute myelomonocytic leukemia by interphase fluorescence in situ hybridization].

OBJECTIVE: To evaluate fluorescence in situ hybridization (FISH) in the detection of inv (16) (p13; q22). METHODS: Spectrum red labeled yeast artificial chromosome (YAC) clone 854E2 which spans the breakpoint cluster region in MYH11 in band 16p13 and single color interphase FISH were used to detect inv (16) in 26 cases of acute myelomonocytic leukemias (AML-M(4)), and the results were compared with that of conventional cytogenetic analysis. RESULTS: R banding karyotyping test revealed no inv (16) in 25 cases, one AML M(4Eo) case showed inv (16) by G banding. Nine cases including all three M(4Eo) had inv (16) by FISH analysis, among whom the characteristic fluorescence signal pattern of the inv (16) was seen in 13.3% to 32.1% (median, 21.3%) of the tested cells. CONCLUSION: YAC 854E2 and interphase FISH provide a powerful technique in the detection of inv (16) (p13q22).

Adult↗
PubMedDate not supplied

Interphase death in human peripheral blood lymphocytes after moderate and high doses of low and high LET radiation: an electron microscopic approach.

Peripheral blood lymphocytes irradiated with low and moderate doses of low linear energy transfer (LET) gamma-rays are known to die by an apoptotic process. In the present study, the type of interphase death occuring after administration of moderate and high doses of low LET gamma-rays and high LET fast neutrons was investigated. Lymphocytes were irradiated in vitro with radiation doses of 5 and 20 gray (Gy) of both radiation qualities. They were cultured for 24 or 48 hours and the type of cell death induced was determined by electron microscopy. After neutron irradiation, a slight increase in the incidence of apoptosis from 5 to 20 Gy was found, whereas after gamma-irradiation, the incidence of apoptosis was lower at 20 Gy as compared to 5 Gy. However, unlike the other radiation doses, the 20 Gy dose of gamma-rays, besides apoptosis also induced oncosis (classical necrosis). According to our experiments, membranes are probably an important target for the induction of interphase death. It is suggested that a great amount of ionisations distributed all over the cell surface, as caused by high doses of gamma-rays, lead to a high influx of Ca++ which induces oncosis instead of apoptosis.

Cell Death↗
PubMed2003

Interphase cytogenetic abnormalities in chronic lymphocytic leukemia may predict response to rituximab.

Select cytogenetic abnormalities such as del(17)(p13.1) and del(11)(q22-q23)predict rapid disease progression and inferior survival in chronic lymphocytic leukemia (CLL). We sought to determine the impact of the four most common interphase cytogenetic abnormalities in 28 CLL patients relative to response to three-times-a-week rituximab therapy. Abnormalities were noted in 25 of the 28 patients to include del(13)(q14.3) [n = 16 (57%)], del(11)(q22.3) [n = 10 (36%)], +12 [n = 6 (21%)], del(17)(p13.1) [n = 5 (18%)], and normal [n = 3 (11%)]. Only a minority of each of these occurred as sole abnormalities. To categorize patients into one specific group, we used the hierarchical order del(17)(p13.1) > del(11)(q22.3) > trisomy 12 > del(13)(q14.3) to prioritize. Response to rituximab was noted to vary by cytogenetic group: del(17)(p13.1), 0% [n = 5]; del(11)(q22.3), 66% [n = 9]; del(13)(q14.3), 86% [n = 7]; +12, 25% [n = 4], and normal, 0% [n = 3]. Response was significantly lower (P = 0.05) in patients with del(17)(p13.1) as compared with those with other abnormalities. These data suggest that interphase cytogenetics in CLL may be predictive of a response to rituximab therapy and provide support for additional studies validating risk-adapted therapy in this disease.

Antibodies, Monoclonal↗
PubMed2003

Interphase fluorescence in situ hybridization assay for the detection of rearrangements of the EVI-1 locus in chromosome band 3q26 in myeloid malignancies.

BACKGROUND AND OBJECTIVES: Rearrangements of the EVI-1 locus in chromosome band 3q26 are associated with a poor prognosis in myeloid malignancies. To aid the diagnosis of such aberrations, and possibly disease monitoring, we established an interphase fluorescence in situ hybridization (FISH) assay for the affected breakpoint region. DESIGN AND METHODS: Several overlapping PAC (P1-derived artificial chromosome) clones centromeric to the EVI-1 gene were labeled with a red fluorescent dye, and PAC clones telomeric to EVI-1 with a green fluorochrome. This dual-color probe was hybridized to cytogenetic preparations of cell lines and patients' samples, which were also investigated for the presence of 3q26 rearrangements by chromosome banding analysis. RESULTS: In nuclei without 3q26 rearrangements, two pairs of co-localized red and green signals were observed, while separation of one red/green signal pair or splitting of one red or one green signal was found when 3q26 aberrations were present. The threshold value for true positivity, as determined on 20 samples from patients with myeloid malignancies without 3q26 rearrangements, was 10.2% for separation of one red/green signal pair, and 1% and 1.3% for splitting of one red or one green signal, respectively. In 17 samples from patients with a 3q26 aberration, the percentage of aberrant cells was significantly above these threshold levels. INTERPRETATION AND CONCLUSIONS: We established an interphase FISH assay that efficiently identifies chromosome breakpoints affecting the EVI-1 locus in 3q26, and represents a useful complement to chromosome banding analysis for the detection of such aberrations.

Acute Disease↗
PubMed2002

Molecular cytogenetics in metaphase and interphase cells for cancer and genetic research, diagnosis and prognosis. Application in tissue sections and cell suspensions.

As the pioneer among molecular cytogenetics techniques, fluorescence in situ hybridization (FISH) allows identification of specific sequences in a structurally preserved cell, in metaphase or interphase. This technique, based on the complementary double-stranded nature of DNA, hybridizes labeled specific DNA (probe). The probe, bound to the target, will be developed into a fluorescent signal. The fact that the signal can be detected clearly, even when fixed in interphase, improves the accuracy of the results, since in some cases it is extremely difficult to obtain mitotic samples. FISH is still used mostly in research, but there are diagnostic applications. New nomenclature is being developed in order to define many of the aberrations that were not distinguished before FISH. Prenatal diagnosis of aneuploidies and malignancies are promptly detected with FISH, which is very useful in critical cases. In some tumors, where chromosomal abnormalities are too complicated to classify manually, the technique of comparative genomic hybridization (CGH), a competitive FISH, allows examiners to determine complete or partial gain or loss of chromosomes. CGH results allow the classification of many tumor cell lines and along with other complementary techniques, like microdissection-FISH, PRINS, etc., increase the possibility of choosing an appropriate treatment for cancer patients.

Chromosome Aberrations↗
PubMed1992

Interphase detection of trisomy 12 in B-cell chronic lymphocytic leukemia by fluorescence hybridization in situ.

Fluorescence hybridization in situ with a probe specific for the alphoid sequence of the centromeric region of chromosome 12 was used to evaluate seven cases of B-cell chronic lymphocytic leukemia (B-CLL). Trisomy 12 was detected in interphase cells from one case which did not produce analyzable metaphases. Four out of the seven cases studied showed clear evidence of trisomy 12 in interphase nuclei. In situ hybridization provides an important supplement to classic cytogenetic studies of B-CLL and may help refine our understanding of the clinical correlations of chromosome abnormalities.

Adult↗
PubMed1992

Induction by H2O2 of DNA and interphase chromosome damage in plateau-phase Chinese hamster ovary cells.

The induction by H2O2 of DNA breaks, DNA double-strand breaks (DSBs), and interphase chromatin damage and their relationship to cytotoxicity were studied in plateau-phase Chinese hamster ovary (CHO) cells. Damage in interphase chromatin was assayed by means of premature chromosome condensation (PCC); DNA DSBs were assayed by nondenaturing filter elution (pH 9.6), and DNA breaks by hydroxyapatite chromatography. Cells were treated with H2O2 in suspension at 0 degrees C for 30 min and treatment was terminated by the addition of catalase. Concentrations of H2O2 lower than 1 mM were not cytotoxic, whereas concentrations of 40 and 60 mM reduced cell survival to 0.1 and 0.004, respectively. An induction of DNA breaks that was dependent on H2O2 concentration was observed at low H2O2 concentrations that reached a maximum at approximately 1 mM; at higher H2O2 concentrations induction of DNA breaks either remained unchanged or decreased. Damage at the chromosome level was not evenly distributed among the cells, when compared to that expected based on a Poisson distribution. Three categories of cells were identified after exposure to H2O2: cells with intact, control-like chromosomes, cells showing chromosome fragmentation similar to that observed in cells exposed to ionizing radiation, and cells showing a loss in the ability of their chromatin to condense into chromosomes under the PCC reaction. The fraction of cells with fragmented chromosomes, as well as the number of excess chromosomes per cell, showed a dose response similar to that of DNA DSBs, reaching a maximum at 1 mM and decreasing at higher concentrations. The results indicate that induction of DNA and chromosome damage by H2O2 follows a complex dependence probably resulting from a depletion of reducing equivalents in the vicinity of the DNA. Reducing equivalents are required to recycle the transition metal ions that are needed to maintain a Fenton-type reaction. The absence of cell killing at H2O2 concentrations that yielded the maximum amount of DNA and chromosome damage suggests that this damage is nonlethal and repairable. It is suggested that lethal DNA and chromosome damage is induced at higher concentrations of H2O2 where cell killing is observed by an unidentified mechanism.

Animals↗
PubMed2005

[Differentiation of interphase domains of the homologous human X-chromosomes].

In accordance with molecular biology data reported elsewhere, homologous interphase X-chromosome territories differ greatly in the abundance of inactive condensed chromatin. On the other hand, a three dimensional FISH (3D FISH) method has revealed that domains of both inactive and active X-chromosome have similar volumes and similar maximum section areas (Smax). To solve this contradiction, we examined differences between homologous human interphase X-chromosome territories using two dimensional FISH (2D FISH) preparations of clustered PHA-stimulated lymphocytes. For obtaining such preparations, we developed a new technique to avoid a stage of hypotonic treatment of living cells, since this treatment levels the chromatin compactness degree. According to our 2D FISH data, the mean ratios of Smax for larger and smaller homologous X-chromosomes, calculated for individual flattened nuclei, were 1.83 +/- 0.08 and 2.02 +/- 0.09, respectively, for clumped cells and groups of loosely associated and separated lymphocytes. In comparison, the same ratio calculated for individual 3D nuclei of PHA-stimulated lymphocytes was 1.38 +/- 0.05 (Falk et al., 2002). Our findings give evidence for enrichment of inactive X-chromosomes by low stretchable condensed chromatin. In addition, these findings show that an active X is enriched by a high stretchable form of chromatin, whose content may distinctly vary from cell to cell.

Cells, Cultured↗
PubMed2006

[Differentiation of interphase nucleolus organizers in embryonic pig kidney cells (PK cell line)].

The prometaphase karyotype of cell line PK contains two heteromorphous pairs of nucleolus organizers that belong to chromosomes 8a and 8, and to 10L and 10s. It was proposed that such heteromorphism may promote chromosome differentiating of interphase nucleolus organizers (INOs) with linear configuration. To test this assumption, we used two-dimensional (2D) preparations of methanol fixed PK cells surface stretched without hypotonic treatment. It was shown that in these preparations the large bulk of interphase PK cells contained 3-4 necklace-like linear structures arranged in nucleolar domains. The observed structures were positive in phase contrast and after DAPI-staining. Complimentary rDNA-FISH revealed that these structures were INOs, the largest iNO in individual cells containing prominent terminal rDNA FISH/DAPI signal. In accordance with the data on prometaphase analysis, the latter INOs belong to chromosomes 8a. As reported by Smetana and coworkers (1999), proteins of the nucleolar fibrillar center reacted preferentially with silver in methanol fixed unwashed smears of human peripheral lymphocytes. It was established that the same specific silver reaction is characteristic most probably of 2D preparations of methanol fixed PK cells. Both silver stained and rDNA-FISH linearized INOs had necklace-like or banded structure with different degrees of resolution. Banded INOs consisted of transverse argyrophilic structures: dense bands and loose interbands. High resolved banded INOs revealed a longitudinal splitting (binemic structure) of interband zone. Necklace-like INOs consisted of argyrophilic beads nearly two-fold more narrow than argyrophilic bands, and uninemic or silver-negative interbead zones. Our findings evidence that necklace-like INOs are typical for G1 and S phase cells, whereas banded INOs are characteristic of G2 cells. Among high resolved linear INOs, we found four reproducible patterns of silver staining, which could be combined it two homologous groups. Because each given pattern is unique for individual PK cells, we concluded that the patterns under study were chromosome specific. Using prometaphase analysis data, we determined chromosome affiliation for each of the four tested patterns of INO silver staining. High resolved INOs, belonging to different chromosomes, were further compared with regard to their average length and the mean of argyrophilic bead number per individual INO, in addition to the length and argyrophilic bead number ratios calculated for different INO pairs of individual cells. Surprisingly, we found that both the ratios, detected for most heteromorphous pair of homologous chromosomes 8a and 8, made only 1.26 +/- 0.02. In comparison, the similar length ratio for nucleolus organizers in chromosomes 8a and 8, calculated for individual prometaphase cells, reached 2.92 +/- 0.30.

Animals↗
PubMed1991

Interphase cytogenetic analysis in the diagnosis and study of neoplastic disorders.

Cytogenetic information usually is obtained through the direct analysis of chromosomes from cells arrested in metaphase. Recently, advances in molecular genetics have made it possible to acquire cytogenetic information through the study of interphase and terminally differentiated cells. By using chromosome-specific DNA probes or probes that are specific for certain chromosomal regions, and by employing techniques of in situ hybridization along with nonradioactive detection methods, it is now possible to detect numerical and structural chromosomal abnormalities from nonmetaphase cells. When used as an adjunct to conventional cytogenetic analysis or when used together with knowledge of established cytogenetic findings for a particular malignancy, this new technology can serve to broaden the scope and utility of cytogenetic analysis beyond the limits of the present metaphase-based technology. Interphase cytogenetic analysis has application to the diagnosis and study of neoplastic disorders and, thus, has particular importance in pathology.

Chromosome Aberrations↗
PubMed1990

Immunoaffinity purification and functional characterization of interphase and meiotic Drosophila nuclear lamin isoforms.

Three isoforms of a single nuclear lamin have been identified in Drosophila. Two, lamins Dm1 and Dm2, are present during interphase and are apparently in equilibrium with each other in vivo. The third, lamin Dmmit, is found in cells that have undergone nuclear envelope breakdown, either during meiosis or mitosis. All three isoforms were purified under nondenaturing conditions using a novel technique of immunoaffinity chromatography and their in vitro activities were examined. Interphase lamins Dm1 and Dm2 can assemble into filaments at physiologic ionic strength; assembly is reversible upon addition of concentrated NaCl. Negative staining of filaments formed in vitro shows long, unbranched bundles approximately 20 nm in diameter. Addition of specific antilamin antibodies blocks in vitro assembly completely. In contrast with lamins Dm1 and Dm2, lamin Dmmit remains soluble at physiologic ionic strength. These observations are consistent with the notion that lamina disassembly in vivo is due, at least in part, to changes in properties of the lamins themselves.

Animals↗
PubMed1990

[The interphase chromatin of lymphocyte nuclei in psoriasis patients].

Lymphocytic nuclei interphase chromatin was examined with the use of automated morphometry in psoriasis patients treated by different methods. Fifty optic and geometric parameters of lymphocytes were under study; the nuclear size and relative contrast, relative contrast of nongranular component, and chromatin pattern determined visually were found the most informative characteristics. The results evidence significant differences between the reference group of normal subjects and psoriasis patients. After treatment a tendency to normalization of the shifted parameters of interphase chromatin can be traced; PUVA therapy intensifies this process, and the parameters reach the reference values.

Adult↗
PubMed1989

[A quantitative study of Ag-positive nucleolar segments revealed by silver staining in the interphase nuclei of trophoblast cells from the rat placental connective zone].

A cytomorphological study was made of silver stained nucleoli in interphasic nuclei of trophoblast cells from the rat placenta connective zone, in addition to calculation of Ag-positive spherules in the nucleoli. The prevalent number of Ag-positive nucleolar spherules in the nuclei was 6, corresponding to the number of nucleolar organizers (NOR's) in the diploid chromosome complement of the rat. The mean number of Ag-positive spherules in the nucleoli progressively increase in the course of polyploidization from 2c to 32c; variability of the spherule number also increasing. The mean area of nucleoli is found to increase in proportion to the ploidy degree. A high correlation is found between the number of Ag-positive spherules and the area of nucleoli in the nucleus (r = 0.78). This appropriateness is exhibited at all the ploidy levels. The number of Ag-spherules and the area of nucleoli are found to depend slightly on the number of nucleoli. The possibility to use the number of Ag-positive spherules as a criterion of the activity of the NOR in interphasic nuclei is discussed.

Animals↗