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PubMed2002

Ultra-rapid multicolor PRINS protocol for chromosome detection in human sperm.

We report a new multicolor PRINS procedure for chromosome identification on human sperm. Based on the direct in-situ mixing of the colors of the fluorochromes (FITC, TRITC, Cascade Blue) incorporated in sequential PRINS reactions, this method facilitates rapid distinct labeling of 3 or 4 chromosomes. Each PRINS reaction consists of a unique 4 minute step for annealing and elongation. The method was successfully tested on lymphocytes and spermatozoa. Estimates of disomy were performed for chromosomes 7, 9 and 16 on sperm samples from 2 healthy donors. There was no significant difference between the disomy rates obtained with the conventional two-color PRINS technique and this new three-color procedure. By simplifying the multicolor PRINS protocol, this new protocol should facilitate the use and adaptation of PRINS to various cytogenetic applications.

Chromosomes, Human, Pair 16↗
PubMed2001

Limitations of chromosome classification by multicolor karyotyping.

Multicolor karyotyping technologies, such as spectral karyotyping (SKY) (Schröck et al.1996; Liyanage et al. 1996) and multiplex (M-) FISH (Speicher et al. 1996), have proved to be extremely useful in prenatal, postnatal, and cancer cytogenetics. However, these technologies have inherent limitations that, in certain situations, may result in chromosomal misclassification. In this report, we present nine cases, which fall into five categories, in which multicolor karyotyping has produced erroneous interpretations. Most errors appear to have a similar mechanistic basis.

Animals↗
PubMed1997

Meiotic segregation, recombination, and gamete aneuploidy assessed in a t(1;10)(p22.1;q22.3) reciprocal translocation carrier by three- and four-probe multicolor FISH in sperm.

Meiotic segregation, recombination, and aneuploidy was assessed for sperm from a t(1;10)(p22.1;q22.3) reciprocal translocation carrier, by use of two multicolor FISH methods. The first method utilized three DNA probes (a telomeric and a centromeric probe on chromosome 1 plus a centromeric probe on chromosome 10) to analyze segregation patterns, in sperm, of the chromosomes involved in the translocation. The aggregate frequency of sperm products from alternate and adjacent I segregation was 90.5%, and the total frequency of normal and chromosomally balanced sperm was 48.1%. The frequencies of sperm products from adjacent II segregation and from 3:1 segregation were 4.9% and 3.9%, respectively. Reciprocal sperm products from adjacent I segregation deviated significantly from the expected 1:1 ratio (P < .0001). Our assay allowed us to evaluate recombination events in the interstitial segments at adjacent II segregation. The frequencies of sperm products resulting from interstitial recombination in chromosome 10 were significantly higher than those resulting from interstitial recombination in chromosome 1 (P < .006). No evidence of an interchromosomal effect on aneuploidy was found by use of a second FISH method that simultaneously utilized four chromosome-specific DNA probes to quantify the frequencies of aneuploid sperm for chromosomes X, Y, 18, and 21. However, a significant higher frequency of diploid sperm was detected in the translocation carrier than was detected in chromosomally normal and healthy controls. This study illustrates the advantages of multicolor FISH for assessment of the reproductive risk associated with translocation carriers and for investigation of the mechanisms of meiotic segregation of chromosomes.

Adult↗
PubMed1999

Detection of aneuploidy by multicolor FISH in mouse sperm after in vivo treatment with acrylamide, colchicine, diazepam or thiabendazole.

Multicolor fluorescence in situ hybridization (FISH) was used to investigate the induction of aneuploidy during meiosis in young adult male mice treated with chemicals chosen for the EU sponsored aneuploidy project (acrylamide, colchicine, diazepam and thiabendazole). The aim of the present study was to evaluate the frequency of aneuploid sperm induced by each of these chemicals by sperm FISH. Male (102/ElxC3H/El)F1 mice were treated with acrylamide (120 and 60 mg/kg single dose i.p.), colchicine (1.5 and 3 mg/kg single dose, i.p.), diazepam (300, 150 and 75 mg/kg single dose by oral intubation) or thiabendazole (100 and 300 mg/kg daily for 11 days by oral intubation). At 22 days after the last treatment, sperm were collected from the cauda epididymis. Three chromosome FISH was applied to determine hyperhaploid and diploid sperm with DNA probes specific for the chromosomes X, Y and 8. Five animals were treated per dose group and sperm aneuploidy was evaluated in 10,000 sperm per animal. We found significant increases in the frequency of total hyperhaploidy for the males treated with 3.0 mg/kg colchicine (0.092 versus 0.056%, P < 0.05) and with 1.5 mg/kg colchicine (0.082 versus 0.050%, P < 0.05), as well for the males treated with 300 mg/kg diazepam (0.081 versus 0.050%, P < 0.05), indicating that colchicine and diazepam each induced germ cell aneuploidy. We also found significant increases in the frequency of total diploidy for the males treated with 300 mg/kg diazepam (P < 0.05) and with 300 mg/kg thiabendazole (P < 0.05). No significant effects were found for 120 and 60 mg/kg acrylamide or for the other doses of diazepam and thiabendazole. These first results indicate that the multicolor FISH method is useful to determine aneuploidy induction in sperm of mice.

Acrylamide↗
PubMed1996

Multicolor fluorescence in situ hybridization analysis of aneuploidy and diploidy frequencies in 225,846 sperm from 10 normal men.

Aneuploidy and diploidy frequencies for chromosomes 1, 12, X, and Y were assessed in 225,846 sperm from 10 normal men. Results from 5 of the men have previously been reported. Multicolor fluorescence in situ hybridization (FISH) was used to control for lack of probe hybridization and to distinguish diploidy from disomy. A minimum of 10,000 sperm per donor were evaluated for each chromosome. Sperm were considered disomic if two fluorescent signals were separated by a distance of a minimum of one signal domain. The mean frequencies of disomic sperm for chromosomes 1 and 12 were 0.11% (range 0.05-0.18%) and 0.16% (range 0.10-0.25%), respectively. The means for the sex chromosomal aneuploidies were 0.07% XX, 0.18% YY, and 0.16% XY, totaling 0.42% for all sex chromosomes (range 0.23-0.71%). The incidence of disomic sperm for the sex chromosomes was significantly increased compared to the frequency for the autosomes, corroborating results obtained from studies of sperm karyotypes and spontaneous abortions. The mean frequencies of single X- and Y-bearing sperm were 50.1% and 49.0%, respectively--not significantly different from 50%. The mean frequency of diploid sperm was 0.16% (0.06-0.42%). Interdonor heterogeneity was found to exist for disomy 1, XX, YY, and diploidy, suggesting significant variation among normal men. Comparison of these FISH results to our historical sperm karyotypes demonstrated that the sex ratios and disomy frequencies for chromosomes 1 and X were similar. However, there was a significantly increased frequency of disomic sperm for chromosomes 12, YY, and XY in FISH data compared with sperm karyotypes. In general, FISH data agreed quite well with values from sperm karyotyping, including the increased frequency of sex chromosomal aneuploidy compared with autosomal aneuploidy in sperm. Multicolor FISH analysis permits an accurate distinction between disomic and diploid sperm and allows analysis of large sample sizes. This powerful technology may be useful for future studies of potential environmental and occupational mutagens.

Adult↗
PubMed2003

Characterization of six wheat x Thinopyrum intermedium derivatives by GISH, RFLP, and multicolor GISH.

Restriction fragment length polymorphism (RFLP) analysis and multicolor genomic in situ hybridization (GISH) are useful tools to precisely characterize genetic stocks derived from crosses of wheat (Triticum aestivum) with Thinopyrum intermedium and Thinopyrum elongatum. The wheat x Th. intermedium derived stocks designated Z1, Z2, Z3, Z4, Z5, and Z6 were initially screened by multicolor GISH using Aegilops speltoides genomic DNA for blocking and various combinations of genomic DNA from Th. intermedium, Triticum urartu, and Aegilops tauschii for probes. The probing (GISH) results indicated that lines Z1 and Z3 were alien disomic addition lines with chromosome numbers of 2n = 44. Z2 was a substitution line in which chromosome 2D was substituted by a pair of Th. intermedium chromosomes; this was confirmed by RFLP and muticolour GISH. Z4 (2n = 44) contained two pairs of wheat--Th. intermedium translocated chromosomes; one pair involved A-genome chromosomes, the other involved D- and A- genome chromosomes. Z5 (2n = 44) contained one pair of wheat--Th. intermedium translocated chromosomes involving the D- and A-genome chromosomes of wheat. Z6 (2n = 44) contained one pair of chromosomes derived from Th. intermedium plus another pair of translocated chromosomes involving B-genome chromosomes of wheat Line Z2 was of special interest because it has some resistance to infection by Fusarium graminearum.

Blotting, Southern↗
PubMed1999

High resolution multicolor-banding: a new technique for refined FISH analysis of human chromosomes.

A new multicolor-banding technique has been developed which allows the differentiation of chromosome region specific areas at the band level. This technique is based on the use of differently labeled overlapping microdissection libraries. The changing fluorescence intensity ratios along the chromosomes are used to assign different pseudo-colors to specific chromosome regions. The multicolor banding of human chromosome 5 is presented as an example.

Chromosome Banding↗
PubMed2002

Direct demonstration of the productive capability of cytokines at the single cell level in lung sarcoidosis using multicolor cytometry.

BACKGROUND: Sarcoidosis is a chronic systemic disorder of unknown etiology characterized by noncaseating epithelioid cell granulomatous lesions, around which an increasing number of CD4+ T cells infiltrate. These CD4+ T cells may release interferon-gamma (IFN-gamma) and interleukin-2 (IL-2). These cytokines are considered to play an important role in pathogenesis of sarcoidosis. METHODS: We employed a modification of Jung's method using multicolor flow cytometry to assess the capability of single cells obtained from bronchoalveolar lavage (BAL) fluid to produce various cytokines. BAL CD4+ T cell production of IFN-gamma, IL-2 and IL-4 after phorbol ester and ionomycin stimulation were studied. RESULTS: The percentage of IFN-gamma- and IL-2-producing CD4+ T cells was significantly higher in patients with sarcoidosis compared to healthy volunteers [84.7 +/- 7.5 vs. 51.2 +/- 14.8% (p < 0.005), and 75.3 +/- 8.7 vs. 39.8 +/-11.0% (p < 0.001), respectively]. No significant difference in the percentage of IL-4-producing CD4+ T cells was noted (1.2 +/- 0.6 vs. 3.5 +/- 2.6%; not significant), whereas the absolute number of IL-4-producing CD4+ T cells was significantly higher in patients with sarcoidosis compared to healthy volunteers (563.6 +/- 330.2 vs. 50.9 +/- 66.9/ml; p < 0.005). In the IL-4-producing CD4+ T cells, about 80% of cells concomitantly produced IFN-gamma and more than 60% of cells also produced IL-2. CONCLUSION: We demonstrate that Th1-like-producing cells are predominant in the CD4+ as well as in the CD8+ T cell subset of patients with sarcoidosis. We for the first time demonstrated concomitant capabilities of BAL CD4+ T cells to produce Th1 and Th2 cytokines at the single cell level by multicolor flow cytometry.

Adult↗
PubMed2006

Prism-based multicolor fluorescence correlation spectrometer.

We report the design and application of a prism-based detection system for fluorescence (cross) correlation spectroscopy. The system utilizes a single laser wavelength for the simultaneous excitation of several dyes of different emission spectra. Fluorescence light is spectrally separated with a prismatic setup, and wavelengths are selected by scanning a fiber-coupled avalanche photodiode across the image spots. Multicolor autocorrelations are demonstrated with standard and tandem dyes, and fluorescence cross-correlation measurements of biotinylated nanocontainers and streptavidin are presented. This spectrometer offers high optical stability and no focal volume mismatch for the multicolor detection of molecular dynamics and interactions, with single-molecule sensitivity.

Equipment Design↗
PubMed2002

Enhanced formation of extracellular laccase activity by the white-rot fungus Trametes multicolor.

The white-rot fungus Trametes multicolor MB 49 has been identified as an excellent producer of the industrially important enzyme laccase. The formation of extracellular laccase could be considerably stimulated by the addition of Cu(II) to a simple, glycerol-based culture medium. In this study, optimal concentrations of copper were found to be 0.5-1 mM, which were added during the growth phase of the fungus. Other medium components important for laccase production are the carbon and nitrogen sources employed. When using an optimized medium containing glycerol (40 g/L), peptone from meat (15 g/L), and MgSO4 x 7H2O and stimulating enzyme formation by the addition of 1.0 mM Cu, maximal laccase activities obtained in shake-flask cultures were approx 85 U/mL. These results, however, could not be scaled up to a laboratory fermentor cultivation. Laccase production by T. multicolor decreased considerably when the fungus was grown in a stirred-tank reactor, presumably because of damage of the mycelia caused by shear stress and/or changes in the morphology of the fungus.

Ascomycota↗
PubMed2006

Single-step multicolor fluorescence in situ hybridization using semiconductor quantum dot-DNA conjugates.

We report a rapid method for the direct multicolor imaging of multiple subnuclear genetic sequences using novel quantum dot-based fluorescence in situ hybridization (FISH) probes (QD-FISH). Short DNA oligonucleotides were attached on QDs and used in a single hybridization/detection step of target sites in situ. QD-FISH probes penetrate both intact interphase nuclei and metaphase chromosomes and showed good targeting of dense chromatin domains with minimal steric hindrances. We further demonstrated that QD's broad absorption spectra allowed different colored probes specific for distinct subnuclear genetic sequences to be simultaneously excited with a single excitation wavelength and imaged free of chromatic aberrations in a single exposure. Thus, these results demonstrate that QD-FISH probes are very effective in multicolor FISH applications. This work also documents new possibilities of using QD-FISH probes detection down to the single molecule level.

Animals↗
PubMed2004

Multicolor FISH probe sets and their applications.

Multicolor fluorescence in situ hybridization (FISH) assays are nowadays indispensable for a precise description of complex chromosomal rearrangements. Routine application of such techniques on human chromosomes started in 1996 with the simultaneous use of all 24 human whole chromosome painting probes in multiplex-FISH (M-FISH) and spectral karyotyping (SKY). Since then different approaches for chromosomal differentiation based on multicolor-FISH (mFISH) assays have been described. Predominantly, they have been established to characterize marker chromosomes identified in conventional banding analysis. Their characterization is of high clinical impact and is the requisite condition for further molecular investigations aimed at the identification of disease-related genes. Here we present a review on the available mFISH methods including their advantages, limitations and possible applications.

Animals↗
PubMed2004

Chromosome intrachanges and interchanges detected by multicolor banding in lymphocytes: searching for clastogen signatures in the human genome.

Genomic fingerprints of mutagenic agents would have wide applications in the field of cancer biology, epidemiology and prevention. The differential spectra of chromosomal aberrations induced by different clastogens suggest that ratios of specific aberrations can be exploited as biomarkers of carcinogen exposure. We have tested this hypothesis using the novel technique of multicolor banding in situ hybridization (mBAND) in human peripheral blood lymphocytes exposed in vitro to X rays, neutrons, heavy ions, or the restriction endonuclease AluI. In the heavy-ion-irradiated cells, we further analyzed aberrations in chromosome 5 using multicolor FISH (mFISH). Contrary to the expectations of biophysical models, our results do not support the use of the ratios of inter-/intrachromosomal exchanges or intra-/interarm intrachanges as fingerprints of exposure to densely ionizing radiation. However, our data point to measurable differences in the ratio of complex/simple interchanges after exposure to different clastogens. These data should be considered in current biophysical models of radiation action in living cells.

Biomarkers↗
PubMedDate not supplied

[Conventional and multicolor FICTION as tools for the interdisciplinar study of hematological neoplasms].

Interdisciplinary technologies allowing a comprehensive characterization of cancer cells are becoming increasingly important in the routine diagnostics and research of hematological neoplasms, and in malignancies in general. One of those methods is the FICTION technique (Fluorescence Immunophenotyping and Interphase Cytogenetics as a Tool for the Investigation of Neoplasms). This technique allows the simultaneous study of the basic morphology as well as immunophenotypic and genetic markers of tumor cells. However, the low number of fluorescent substances utilized in a single assay restricts the number of markers that can be studied by FICTION. Recently, a multicolor variant has been developed and termed M-FICTION which in addition to cellular morphology and immunophenotype allows the investigation of multiple chromosomal aberrations in only one assay. In the present review, we present a detailed overview on the conventional and multicolor FICTION techniques and we discuss their multiple applications in the field of hematological neoplasms.

English Abstract↗
PubMed2001

Standardized multicolored magnetic resonance images of gynecologic lesions.

PURPOSE: The purpose of this study was to develop a convenient and simple method for visualizing in color the features of gynecologic lesions by combining T1- and T2-weighted magnetic resonance images using RGB (red-green-blue) color channels. MATERIALS AND METHODS: T1- and T2-weighted film images of gynecologic lesions were digitized using a film-scanner. The signal intensities of fat on T1-weighted images were converted to 133% of those on T2-weighted images. Additive multicolored images in the RGB color system were produced from T1- and T2-weighted images using color axes of a complementary color pair for a total of 84 typical gynecological lesions. RESULTS: Tissues were displayed in color as follows: fat: light tan; urine: light blue: muscle: dark brown; endometrium: light blue; leiomyomas with cystic degeneration: cyanblue; cellular leiomyoma: yellowish brown; endometrial cyst: orange. The images of female intrapelvic structures and gynecologic lesions were semi-natural in appearance. CONCLUSION: Standardization of fat intensities provides a simple method for multicolored visualization of the features of gynecologic lesions.

Color↗
PubMed2000

[Studies on micronuclei induced by colchicine and cyclophosphamide using multicolor fluorescence in situ hybridization].

OBJECTIVE: To study chromosomal composition of micronuclei (MN) induced by colchicine (COL) and cyclophosphamide (CP) in mouse bone marrow erythrocytes. METHODS: Multicolor fluorescence in situ hybridization (FISH) with centromeric and telomeric DNA probes was applied to analyze chromosomal composition of micronuclei induced by COL and CP in mouse bone marrow erythrocytes. RESULTS: About 83.5% of COL-induced MN revealed both centromeric and telomeric signals. Of the CP-induced MN, 74.5% showed telomeric signals only. CONCLUSION: Majority of COL-induced MN contain whole chromosome and that of CP-induced MN mainly contain acentric fragments. Multicolor FISH with centromeric and telomeric DNA probes was a precise technique for analyzing chromosomal composition of MN.

Animals↗
PubMed2004

[Multicolor fluorescence in situ hybridization and its application in genetic toxicology].

Multicolor fluorescence in situ hybridization, a new promising technology today, offers unparalleled capabilities for detection of nucleic acid sequences, chromosomes and genes. Owing to structural and numerical chromosomal aberrations were considered as important biological end points in genotoxic studies, chromosomal aberrations after exposure to occupational, medical, and environmental toxicants could be accurately and appropriately evaluated via this assay. Consequently, multicolor FISH-based methods could be widespread applied in human genetics, reproductive medicine, especially genetic toxicology. In this paper, the progress in regard was reviewed.

Animals↗
PubMed2005

A subset of pancreatic adenocarcinomas demonstrates coamplification of topoisomerase IIalpha and HER2/neu: use of immunolabeling and multicolor FISH for potential patient screening andtreatment.

We sought to identify the frequency of amplification of the topoisomerase IIalpha gene (TOP2A) in pancreatic cancer and determine the usefulness of TOP2A immunolabeling in screening for TOP2A and human epidermal growth factor receptor (HER)2/neu amplification. We examined 55 pancreatic adenocarcinoma specimens for TOP2A immunolabeling and identified TOP2A protein expression in all specimens with a nuclear labeling index (NLI; positive nuclei/total nuclei x 100) of 5% to 80%. Normal pancreatic ductal epithelium, proposed to give rise to pancreatic adenocarcinoma, did not demonstrate detectable TOP2A expression. In a subset of specimens selected for fluorescence in situ hybridization analysis of TOP2A and HER2/neu amplification using a recently developed multicolor probe, 7 of 8 lesions with an NLI of 25% or more demonstrated TOP2A amplification, in contrast with 2 of 14 lesions with a TOP2A NLI of less than 25%. In 8 of 9 TOP2A-amplified cases, coamplification of HER2/neu was present, suggesting a potential relationship between TOP2A and HER2/neu in pancreatic adenocarcinoma. We propose that TOP2A immunolabeling be used in conjunction with a newly developed multicolor probe to screen patients with pancreatic adenocarcinoma to determine the best potential therapeutic modalities, such as TOP2A inhibitors, trastuzumab, or both.

Adenocarcinoma↗