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PubMed2002

[The application of dual fusion interphase fluorescence in situ hybridization probe in detecting minimal residual disease in chronic myeloid leukemia].

OBJECTIVE: To explore the sensitivity, specificity and reproducibility of dual-color and dual-fusion fluorescence in situ hybridization (D-FISH) in detecting the minimal residual disease (MRD) of chronic myeloid leukemia (CML) after treatment. METHODS: MRD were detected in the bone marrow of 8 cases of chronic myeloid leukemia after treatment with interphase fluorescence in situ hybridization. Sensitivity, specificity and reproducibility of D-FISH were compared with those of single-fusion FISH (S-FISH). Furthermore, correlation between the results of D-FISH and RT-PCR was explored. RESULTS: D-FISH was more sensitive and specific than S-FISH and had a good reproducibility. In normal control, the cutoff rates of D-FISH and S-FISH were 0.570% and 5.868% with significant statistic difference. In detection of MRD in 8 cases of CML post transplantation, positive rates of S-FISH and D-FISH were 3/8 (37.5%) and 5/8 (62.5%) respectively. Furthermore, the results of D-FISH had a high correlation with that of RT-PCR. CONCLUSION: In view of the lower false-positive and false-negative results of D-FISH, it may be used in the detection of minimal residual disease in CML after treatment. But further exploration is required for verifying the correlation between the results of D-FISH and the clinical parameters.

Adolescent↗
PubMed2003

[Detection of human/goat xenogeneic models by interphase fluorescence in situ hybridization].

OBJECTIVE: To establish a high sensitive and specific method of interphase fluorescence in situ hybridization (IFISH) to detect the low-frequency human cells in human/goat xenogeneic models. METHODS: Human-specific Y-chromosome satellite DNA CEPY and 17-chromosome satellite DNA p17H8 were used as probes for IFISH. The peripheral blood samples from 2 goats transplanted with human male hematopoietic stem cells (HSC), 1 normal negative goat and 1 normal man were analyzed. The actual FISH efficiency was confirmed by serial dilutions (1/100, 1/500 and 1/1000) of the cell mixture of normal man and normal negative goat. A set of signal scoring criteria was determined to guarantee the stability and reliability of the method. RESULTS: Positive cell (human cell) frequencies were consistent with the established frequencies for the human/goat cell mixture. The average frequencies of positive cells were 98.60% (CEPY) and 100% (p17H8) for normal man, 0 for normal negative goat, 0.23% (CEPY) and 0.11% (p17H8) for human/goat xenogeneic models. The results demonstrated that low-frequency human cells (male cells confirmed by Y-chromosome probe) existed in human/goat xenogeneic models. CONCLUSION: The IFISH developed in this study is of high sensitivity and specificity and can identify the actual frequency of human cells, which offers a direct, sensitive and specific approach to the detection of low-frequency human cells in human/goat xenogeneic models.

Animals↗
PubMed2003

[Application of interphase fluorescence in situ hybridization for determination of the tumor load in chronic myeloid leukemia].

BACKGROUND & OBJECTIVE: It is essential to evaluate the tumor burden accurately in assessing the degree of response of chronic myeloid leukemia for design of treatment model. The aim of this study was to explore the sensitivity and specificity of interphase fluorescence in situ hybridization (I-FISH) technique in monitoring the tumor load of chronic myeloid leukemia (CML) during treatment. METHODS: Ten cases of normal bone marrow cells were examined using I-FISH technique to define the normal cut-off value. Bone marrow cells from 20 CML patients in treatment were assayed with I-FISH, conventional cytogenetic G-banding (CCG) analysis, and reverse transcriptase polymerase chain reaction (RT-PCR). The tumor load was monitored in a quantitative way. RESULTS: The false-positive bone marrow cells were 0.6%-2.0% in control group by I-FISH; the normal cut-off value was 2.45%. Among 20 CML patients after being treated with IFN-alpha or bone marrow transplantation, Ph(+) clone were detected in 9 cases by CGG with positive cell rate ranging from 16.7% to 100%. However, combining with the cutoff value, bcr-abl(+) cells were found in 16/20(80%) cases by I-FISH with positive cell rate ranging from 2.8% to 99.6%. These 16 patients were assayed with RT-PCR, and 13(81.3%) among them showed bcr-abl mRNA positive. CONCLUSION: I-FISH can be used as a sensitive and specific tool to monitor the change of tumor load in CML during treatment; meanwhile, the results acquired from I-FISH would be more accurate than that from CCG and RT-PCR.

Adolescent↗
PubMed2003

[Detection of core-binding factor in acute leukemia with interphase fluorescence in situ hybridization].

OBJECTIVE: To detect the chromosomal abnormalities involving the core-binding factor (CBF) in acute leukemia at initial diagnosis with interphase in situ hybridization (I-FISH) technique, and monitor the minimal residual disease (MRD) after treatment with I-FISH. This study also aim to compare the sensitivity of I-FISH at initial diagnosis with that of conventional G-banding analysis. METHODS: Based on the diagnosis of bone marrow morphology, 15 acute leukemia patients were examined with conventional G-banding and I-FISH techniques. Seven of these patients were monitored for MRD with I-FISH after treatment. RESULTS: On the basis of the false-positive rate acquired from normal subjects, the normal cutoff values of the 3 probes including AML1/ETO translocation probe, MYH11 breakpoint region probe and ETV6/AML1 translocation probe were 4.13%, 1.95% and 2.12% respectively. With conventional G-banding analysis, 40% (6/15) patients were found with chromosomal abnormality involving CBF, including 5 of the 8 M2 patients with t (8;21) and 1 of the 2 M4EO patients with inv (16). No B-ALL cases were identified with t (12;21). With I-FISH, however, 80% (12/15) of the cases were found with genetic abnormality involving CBF, including all the 8 M2 cases with AML1/ETO fusion gene, both of the M4EO cases with CBFbeta/MYH11 and 2 of the 5 B-ALL cases with ETV6/AML1. In the 7 cases monitored for MRD level with I-FISH, 2 M2 cases and 1 B-ALL case were with positive results. CONCLUSIONS: I-FISH is more sensitive than conventional G-binding analysis in detecting the chromosomal abnormalities involving CBF in acute leukemia. At the time of initial diagnosis, combination of the two techniques may lead to more comprehensive and accurate results.

Adolescent↗
PubMed2004

[Study on mixed lineage leukemia gene rearrangement in AML-M4/M5 by interphase fluorescence in situ hybridization].

OBJECTIVE: To explore the value of fluorescence in situ hybridization (FISH) in the detection of mixed lineage leukemia (MLL) rearrangement and to assess the incidence and prognostic significance of MLL gene rearrangement in AML-M4/M5. METHODS: Bone marrow chromosome preparation of 23 cases of acute myeloid leukemia(AML) consisting of 19 cases with M5 and 4 cases with M4 was made using direct method or short-term culture. Karyotypic analysis was carried out by R-banding technique. Dig labeled 11q23 probe which spans the breakpoint cluster region in MLL was used to detect the MLL rearrangement in the above 23 cases. RESULTS: R-banding karyotyping analysis revealed 11q23 translocation in 7 cases, while FISH analysis detected MLL rearrangement in 12 cases including the above 7 cases. CONCLUSION: Interphase FISH was more sensitive in detecting the MLL rearrangement in AML-M4/M5, compared with the conventional cytogenetic method. MLL rearrangement is highly related to AML-M4/M5; it is an indication of poor prognosis.

Adolescent↗
PubMed2004

Analysis of malignancy-associated DNA changes in interphase nuclei of buccal epithelium in persons with breast diseases.

AIM: To study from the point of view of statistical and geometrical theory of pattern recognition, the peculiarities of the distribution of optical density of DNA in the interphase nuclei of mammary buccal epithelium upon pathology. METHODS: Cytogenetic investigation of buccal smears and computer-based image analysis were used. RESULTS: It is shown that in malignant neoplasms of the mammary glands compared with its values in benign, the optical density of DNA in the nuclei of buccal epithelium increase in a range from 0.15 to 0.30 conventional units. The sensitivity of that criterium varied from 80.5% to 96.8%, and specificity was 92.3%. CONCLUSION: The method proposed may be recommended as an additional one for improvement of the diagnosis of mammary pathologies.

Adult↗
PubMedDate not supplied

Interphase FISH for follow-up of Philadelphia chromosome-positive chronic myeloid leukemia treatment.

BACKGROUND: Several attempts have been made to determine whether interphase fluorescence in situ hybridization (I-FISH) on bone marrow or peripheral blood specimens is a good alternative to conventional cytogenetics (CC) in calculating the residual proportion of Philadelphia (Ph) chromosome-positive cells during treatment follow-up of patients with chronic myeloid leukemia. MATERIALS AND METHODS: Nineteen patients were selected for I-FISH follow-up compared to CC. All samples were also classified into 4 groups according to the percentage of residual Ph chromosome-positive metaphases analyzed in CC. I-FISH was performed using the LSI bcr/abl dual ES color probe (Vysis). RESULTS: A high correlation was observed between the frequency of Ph chromosome-positive cells, assessed by CC and I-FISH (p<0.001). A high correlation was found between CC and I-FISH for 12 patients, but not for the remaining 7. Applying the same classification for I-FISH did not show a good relationship between the two techniques (p<0.001). CONCLUSION: Dual color I-FISH is a reliable method to monitor the size of the Ph chromosome-positive clone in bone marrow of treated CML patients. However, it has to be complementary to conventional cytogenetics because it cannot detect the emergence of other chromosomal abnormalities in Ph chromosome-positive or -negative cells.

Bone Marrow Cells↗
PubMed1992

Metaphase and interphase cytogenetics with Alu-PCR-amplified yeast artificial chromosome clones containing the BCR gene and the protooncogenes c-raf-1, c-fms, and c-erbB-2.

A human yeast artificial chromosome (YAC) library was screened by polymerase chain reaction with oligonucleotide primers defined for DNA sequences of the BCR gene and the protooncogenes c-raf-1, c-fms, and c-erbB-2. Alu-PCR-generated human DNA sequences were obtained from the respective YAC clones and used for fluorescence in situ hybridization experiments under suppression conditions. After chromosomal in situ suppression hybridization to GTG-banded human prometaphase chromosomes, seven of nine initially isolated YAC clones yielded strong signals exclusively in the chromosome bands containing the respective genes. Two clones yielded additional signals on other chromosomes and were excluded from further tests. The band-specific YACs were successfully applied to visualize specific structural chromosome aberrations in peripheral blood cells from patients with myelodysplasia exhibiting del(5)(q13q34), chronic myeloid leukemia and acute lymphocytic leukemia with t(9;22)(q34;q11), acute promyelocytic leukemia (M3) with t(15;17)(q22;q21), and in a cell line established from a proband with the constitutional translocation t(3;8)(p14.2;q24). In addition to the analysis of metaphase spreads, we demonstrate the particular usefulness of these YAC clones in combination with whole chromosome painting to analyze specific chromosome aberrations directly in the interphase nucleus.

Base Sequence↗
PubMed2005

[Interphase cytogenetics in estimation of genomic mutations in somatic cells].

The review considers the current state, possibilities, and perspectives of using interphase cytogenetics in the estimation of genomic mutations in human and animal somatic cells for aims of genetic toxicology and genetic instability analysis. Possible mechanisms underlying action of mutagens causing numeric chromosome aberrations are discussed.

Animals↗
PubMed2005

Characterization of chromosome 14 abnormalities by interphase in situ hybridization and comparative genomic hybridization in 124 meningiomas: correlation with clinical, histopathologic, and prognostic features.

We analyzed quantitative chromosome 14 abnormalities in 124 meningiomas by interphase fluorescence in situ hybridization (iFISH) and confirmed the nature of abnormalities by comparative genomic hybridization (CGH). We correlated the abnormalities with clinical, histopathologic, and prognostic factors. Of 124 cases, 50 (40.3%) showed loss (14.5%) or gain (25.8%) of the 14q32 chromosome region by iFISH. Most corresponded to numeric abnormalities: monosomy (12.9%), trisomy (1.6%), or tetrasomy (24.2%); in only 2 cases (1.6%), chromosome 14 loss did not involve the whole chromosome and was restricted to the 14q31-q32 region (confirmed by CGH). Cases with gain or monosomy corresponded more frequently to histologically malignant tumors (P = .009). Patients with monosomy 14/14q-, but not those with gain, more often were male (P = .04) and had a greater incidence of recurrence (P = .003) and shorter relapse-free survival (P = .03). The 2 patients with loss limited to 14q31-q32 had histologically benign tumors and no relapse after more than 5 years' follow-up. Most meningiomas with chromosome 14 abnormalities have numeric changes, with interstitial deletions of 14q31-q32 present in few cases. Of the abnormalities detected, only monosomy 14 showed an adverse prognostic impact.

Adolescent↗
PubMed2005

[Comparison of standard prognostic factors with the deletion of 13q14 detected by interphase fluorescence in situ hybridization on separated and unseparated bone marrow cells in multiple myeloma].

BACKGROUND: Cytogenetic abnormalities of chromosome 13 are emerging as important prognostic factors in multiple myeloma and have been associated with poor prognosis. METHODS AND RESULTS: The occurrence of 13q14 deletion and other standard laboratory parameters were determined in 40 patients with multiple myeloma. We found that interphase fluorescence in situ hybridization using a locus specific probe for RB1 gene on immunomagnetically selected myeloma cells was more sensitive than non selected cells. The 13q14 deletion was found in 10 of 40 (25.0%) of bone marrow samples without cell selection and in 25 of 40 (62.5%) of samples with CD138+ enriched myeloma cells. Negative correlation was found between albumin and the 13q14 deletion in separated (p = 0.003) as well as in cells without selection (p = 0.010). No significant correlation was found in overall survival of separated and unseparated cells (p = 0.830; p = 0.260) and a similar result was obtained for treatment response after transplantation of separated cells (p = 0.520) or non-separated cells (0.190). CONCLUSIONS: Our results confirm that immunomagnetic selection of CD138+ cells increases the probability of detection of the 13q14 deletion in bone marrow samples. The correlation was found between albumin and the 13q14 deletion in both of type of cells.

Bone Marrow Cells↗
PubMed2005

Monitoring the disruption of nuclear envelopes in interphase cells with GFP-beta-galactosidase.

The nuclear envelope of eukaryotic cells provides a barrier separating nucleus from cytoplasm, thereby regulating the exchange of macromolecules between both compartments. However, in cells exposed to severe forms of stress this barrier may break down, resulting in the mixing of nuclear and cytoplasmic contents. We show here that the fusion protein GFP-beta-galactosidase can be used to evaluate the intactness of nuclear envelopes in HeLa cells that have been exposed to heat and oxidative stress. GFP-beta-galactosidase is restricted to the cytoplasm of interphase cells, but enters the nucleus when nuclear membranes are disrupted. For comparison, we have analyzed the barrier function of nuclear membranes with antibodies against lamin B. Treatment of fixed cells with digitonin permeabilizes the plasma membrane, but leaves nuclear envelopes intact. Consequently, after digitonin incubation antibodies to lamin B can bind their antigen only if nuclear membranes are damaged. For various heat and oxidative stress conditions, we have compared the distribution of GFP-beta-galactosidase with the accessibility of lamin B to antibodies. Our results demonstrate that nuclear envelopes are permeable to antibodies whenever GFP-beta-galactosidase enters the nucleus. GFP-beta-galactosidase is therefore a useful tool for evaluating the disintegration of the nuclear envelope and identifying cells in which a mixing of nuclear and cytoplasmic material takes place.

Fluorescent Antibody Technique, Indirect↗
PubMed2006

Validation of an interphase fluorescence in situ hybridization approach for the detection of MLL gene rearrangements and of the MLL/AF9 fusion in acute myeloid leukemia.

To validate a 2-step FISH assay for the identification of the t(9;11)(p22;q23), 96 acute myeloid leukemias were studied by cytogenetic analysis, FISH and molecular biology. After a first FISH step using an MLL probe, 24/27 cases with 11q23 break showed MLL rearrangement. Southern blotting confirmed FISH data. In the second step, 24 cases with MLL rearrangement were studied using MLL and AF9 probes: 17/18 cases with t(9;11) showed MLL/AF9 fusion. In 6 patients with 11q23/MLL rearrangements other than t(9;11), FISH confirmed MLL involvement and excluded AF9 involvement. This is a reliable method for the identification of MLL/AF9 fusion in interphase cells, allowing for a reclassification of cases with suboptimal chromosome morphology. The frequency of deletion surrounding MLL and AF9 breakpoint is low.

Gene Rearrangement↗
PubMed2005

[Scoring criteria of aneuploidy frequency in interphase nuclei by fish-analysis].

Principles and approaches of assessment of numerical chromosome abnormalities in interphase nuclei by fluorescent in situ hybridization technology are reviewed. The authors' own scoring criteria of results of hybridization of centromere-specific DNA-probes with chromosomal targets in somatic cells by dual color FISH-analysis are suggested. Use of these scoring criteria allows to reduce the level of artificial hypoploidy, whose its frequency becomes similar to that of hyperploidy. This fact indicates that chromosomal nondisjunction is a major mechanism of aneuploidy induction, rather than of chromosome loss.

Adult↗
PubMedDate not supplied

[Chromocenters of interphase nuclei in the mouse liver contain satellite DNA].

In isolated interphase mouse liver nuclei after hypotonic treatment only the chromocenters belonging to the pericentromeric heterochromatin remain in dense form while the main mass of a chromatin is completely decondensed. The centromeric nature of these chromocenters is demonstrated by their capability for C-banding and for hybridization with a satellite mouse DNA.

Animals↗
PubMed1991

Cell-cycle dependence of heat-induced interphase death in mouse L5178Y cells.

Cell lysis and eosin staining were observed in L5178Y cells within the first 3 h of post-hyperthermia incubation at 37 degrees C, after which both leveled to a plateau. Lysis and eosin staining were proportional to the severity of heat in asynchronous cells, whereas it was maximum in the most heat-sensitive M phase, intermediate in S, and least in heat-resistant G1 for the same heat treatment. Further, leakage of labeled [3H]thymidine and a decrease in radioactivity retained within heated cells coincided with an increase in eosin staining, indicating that the dye uptake was due to membrane damage. It was presumed that the eosin-stained fraction represented dead cells. The percentage eosin-stained cells reached a plateau, and this level was used to determine survival; when the results were compared with those obtained by the colony formation method, they were identical. By comparing the two survival assay methods we concluded that cell death after hyperthermia in L5178Y cells is mainly by interphase death in all phases of the cell cycle. The reasons for this conclusion are that a reduction in survival could be detected within one generation of L5178Y cells by the eosin staining method, and the survival values obtained by this method were identical to those obtained by the colony formation method.

Acclimatization↗
PubMed1991

[Method of diagnosing aneuploidies using in situ hybridization: analysis of interphase nuclei].

The possibility of determining chromosomal sex using in situ hybridization of X-specific alphoid DNA probe with interphase human nuclei is studied. Total number of nuclei under study being 14806, more than 85 percent of female ones and more than 95 per cent of male ones are shown to contain two and one grain clusters, respectively. The minor nuclear classes, i.e. containing other numbers of grain clusters, may reflect the effects of polyploidization and cluster aggregation resulting from spatial chromosome distribution. Therefore the positive analysis of sex chromosome constitution on the base of the applied method is possible.

Aneuploidy↗
PubMed1990

Movement of interphase Golgi apparatus in fused mammalian cells and its relationship to cytoskeletal elements and rearrangement of nuclei.

Virus-induced Vero cell fusion was used to analyze the rearrangement of Golgi apparatus during the development of syncytia. Individual Golgi apparatus, associated initially with the separate microtubule-organizing centers in the perinuclear area of fused cells, congregated in the center of the syncytia and formed an extended Golgi complex within 3 to 5 h. The relocation of the Golgi apparatus, but not of nuclei, depended on the presence of an intact microtubule network, since both the microtubule depolymerizing drug nocodazole and the microtubule-stabilizing drug taxol interfered with the formation of an extended Golgi complex. Depolymerization of microfilaments with cytochalasin D and the complete collapse of intermediate filaments induced by microinjected monoclonal antibodies against vimentin had no effect on these processes. Cooling cells to 20 degrees C inhibited both congregation of Golgi apparatus and relocation of nuclei. Visualization of the movement of Golgi apparatus labeled in living cells with fluorescent metabolites of C6-NBD-ceramide showed that relocation of the Golgi apparatus was a process in which congregation and coalescence of the intact organelles was seen, rather than dispersal and reassembly of smaller Golgi elements in the center of the polykaryons. Thus, movement of intact Golgi apparatus in fused interphase cells depends on an undisturbed microtubule network and occurs independently of the relocation of nuclei.

Actin Cytoskeleton↗