Detection of aneuploidy and aneuploidy-inducing agents in human lymphocytes using fluorescence in situ hybridization with chromosome-specific DNA probes

The feasibility of utilizing fluorescence in situ hybridization with chromosome-specific DNA probes as the basis of an assay to detect aneuploidy and aneuploidy-inducing agents in interphase human lymphocytes has been investigated. The assay involves counting the number of hybridization regions in interphase cells to determine the number of copies of a specific chromosome of interest, 22,000 interphase nuclei from untreated 72-h lymphocyte cultures were examined following hybridization with probes for chromosomes 1, 7, 9, 17, X or Y. The combined frequencies of nuclei containing 0, 1, 2, 3 and 4 hybridization regions for the various autosomal chromosomes were 0.004, 0.084, 0.909, 0.003 and 0.001, respectively. Based on these frequencies, scoring 1000-2000 cells should allow detection of aneuploid cells with a 0.012 frequency of hyperdiploidy or a 0.11 frequency of hypodiploidy for a specific chromosome of interest (alpha = 0.05, beta = 0.80). This difference in test sensitivity is related to the higher frequency of cells with one apparent spot. A comparison of the ratio of hybridization region to nuclear area in the two-dimensional images used for this analysis indicates that an overlap of the two regions probably accounts for the high frequency of apparent monosomy observed in normal cells. Treatment with the aneuploidy-inducing chemicals, colchicine, vincristine sulfate and diethylstilbestrol resulted in significant dose-related increases in the number of nuclei containing 3 or more hybridization regions. Treatment with the clastogen sodium arsenite produced only a minor increase in apparently hyperdiploid cells whereas treatment with ionizing radiation, another potent clastogen, resulted in a significant increase in nuclei containing multiple hybridization regions. These results suggest that ionizing radiation is an aneuploidy-inducing agent under these conditions although chromosomal breakage within the hybridization region may account for a portion of the increased frequency of nuclei with multiple hybridization regions. These results indicate that the use of fluorescence in situ hybridization with DNA probes is capable of detecting aneuploid cells occurring at relatively low frequencies within a population of cells. Assays based on these techniques should facilitate a more rapid identification of aneuploidy-inducing environmental and therapeutic agents.     

Image cytometry-based detection of aneuploidy by fluorescence in situ hybridization in suspension

Cytogenetic abnormalities are important diagnostic and prognostic criteria for hematologic malignancies. Karyotyping and fluorescence in situ hybridization (FISH) are the conventional methods by which these abnormalities are detected. The sensitivity of these microscopy-based methods is limited by the abundance of the abnormal cells in the samples and therefore these analyses are commonly not applicable to minimal residual disease (MRD) stages. A flow cytometry-based imaging approach was developed to detect chromosomal abnormalities following FISH in suspension (FISH-IS), which enables the automated analysis of several log-magnitude higher number of cells compared with the microscopy-based approaches. This study demonstrates the applicability of FISH-IS for detecting numerical chromosome aberrations, establishes accuracy, and sensitivity of detection compared with conventional FISH, and feasibility to study procured clinical samples of acute myeloid leukemia (AML). Male and female healthy donor peripheral blood mononuclear cells hybridized with combinations of chromosome enumeration probes (CEP) 8, X, and Y served as models for disomy, monosomy, and trisomy. The sensitivity of detection of monosomies and trisomies amongst 20,000 analyzed cells was determined to be 1% with a high level of precision. A high correlation (R(2) = 0.99) with conventional FISH analysis was found based on the parallel analysis of diagnostic samples procured from 10 AML patients with trisomy 8 (+8). Additionally, FISH-IS analysis of samples procured at the time of clinical remission demonstrated the presence of residual +8 cells indicating that this approach may be used to detect MRD and associated chromosomal defects. ? 2012 International Society for Advancement of Cytometry.     

Rapid-prenatal diagnosis through fluorescence in situ hybridization for preventing aneuploidy related birth defects

BACKGROUND AND OBJECTIVE:

Women with high-risk pregnancies are offered prenatal diagnosis through amniocentesis for cytogenetic analysis of fetal cells. The aim of this study was to evaluate the effectiveness of the rapid fluorescence in situ hybridization (FISH) technique for detecting numerical aberrations of chromosomes 13, 21, 18, X and Y in high-risk pregnancies in an Indian scenario.

MATERIALS AND METHODS:

A total of 163 samples were received for a FISH and/or a full karyotype for prenatal diagnosis from high-risk pregnancies. In 116 samples both conventional culture techniques for getting karyotype through G-banding techniques were applied in conjunction to FISH test using the AneuVysion kit (Abbott Molecular, Inc.), following standard recommended protocol to compare the both the techniques in our setup.

RESULTS:

Out of 116 patients, we got 96 normal for the five major chromosome abnormality and seven patients were found to be abnormal (04 trisomy 21, 02 monosomy X, and 01 trisomy 13) and all the FISH results correlated with conventional cytogenetics. To summarize the results of total 163 patients for the major chromosomal abnormalities analyzed by both/or cytogenetics and FISH there were 140 (86%) normal, 9 (6%) cases were abnormal and another 4 (2.5%) cases were suspicious mosaic and 10 (6%) cases of culture failure. The diagnostic detection rate with FISH in 116 patients was 97.5%. There were no false-positive and false-negative autosomal or sex chromosomal results, within our established criteria for reporting FISH signals.

CONCLUSION:

Rapid FISH is a reliable and prompt method for detecting numerical chromosomal aberrations and has now been implemented as a routine diagnostic procedure for detection of fetal aneuploidy in India.     

Reliability of aneuploidy estimates in human sperm: Results of fluorescence in situ hybridization studies using two different scoring criteria

Aneuploidy estimates for chromosomes 1, 12, X, and Y were obtained in human sperm from five donors using multicolor fluorescence in situ hybridization (FISH) analysis. Disomy frequencies were obtained by scoring a minimum of 10,000 sperm for each chromosome probe per donor. This analysis was replicated for two scoring criteria: one used one half of a signal domain as the minimum distance between two signals to be counted as two and thus disomic; the other set one signal domain as the minimum distance between two signals. A total of 120,870 sperm were assessed using one half of a domain as the criterion, and 113,478 were scored using one domain as the criterion. The percentage of disomy for chromosomes 1, 12, X, Y, and XY was 0.18, 0.16, 0.15, 0.19, and 0.25, respectively, using the one-half-domain criterion, and 0.08, 0.17, 0.07, 0.12, and 0.16, respectively, using the one-domain criterion. The percentage of disomy decreased significantly with use of one domain as the minimum distance for signal separation for all chromosomes except for chromosome number 12. These lower disomy frequencies correlated well with frequencies derived from human sperm karyotypes analyzed in our laboratory. This suggests that the fluorescent signals for chromosomes 1, X, and Y split into more than one domain in decondensed interphase sperm, and that the use of the one-half-domain criterion would lead to an overestimate of aneuploidy frequencies. The factors known to affect aneuploidy estimates derived from FISH studies are discussed, and recommendations for stringent scoring criteria are proposed. © 1995 wiley-Liss, Inc.     

Non-disjunction and chromosome loss in gamma-irradiated human lymphocytes: a fluorescence in situ hybridization analysis using centromere-specific probes

Centromere-specific DNA probes for chromosomes 4, 7 and 18 were used to simultaneously analyze chromosome loss, non-disjunction, breaks within the labeled region, and nucleoplasmic bridges induced by gamma rays in binucleated human lymphocytes. The doses used were 0, 1, 2 and 4 Gy, and approximately 1000 cells were scored per dose. Micronucleus frequency increased in a linear-quadratic fashion. For chromosome loss, significant increases were observed at 2 and 4 Gy, whereas for non-disjunction significant increases were observed at 1 Gy; thus non-disjunction allowed us to detect the effects of radiation at a lower dose than chromosome loss. The use of centromere-specific probes allowed discrimination between the clastogenic and aneugenic effects of ionizing radiation. The analysis of chromosome loss, not taking fragmented signals into account, ensures the detection of an aneugenic effect, which was not possible using pancentromeric probes. The frequency of chromosome breakage within the labeled regions was higher in nuclei than in micronuclei, suggesting an increase in the engulfment of chromosomal material by nuclei as a consequence of the presence of cytochalasin B in the cultures. Chromatin filaments connecting main nuclei, the so-called nucleoplasmic bridges, were observed in irradiated samples, and are a manifestation of rearranged chromosomes producing anaphase bridges.     

Aneuploidy detection in human sperm nuclei using fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) was performed on human interphase sperm nuclei to determine the utility of this technique for aneuploidy detection. Repetitive DNA sequences specific for chromosomes 1, 12 and X were biotinylated and hybridized with mature sperm, which had been treated with cetyltrimethylammonium bromide and dithiothreitol to render them accessible to the probes. Detection of bound probe was accomplished with fluoresceinated avidin and antiavidin. For each of the chromosomes studied, chromosome number was determined by counting the fluorescent signals, representing hybridized regions, within the sperm nuclei. The frequencies for disomy, that is for nuclei containing two signals, for chromosomes 1, 12 and X were 0.06%, 0.04% and 0.03%, respectively. The congruence of these results with those determined by the cross-species hamster oocyte-human sperm assay, and the high efficiency of hybridization indicate that FISH is a sensitive and reliable tool for aneuploidy detection in human sperm.     

Identification of individuals at high risk for head and neck carcinogenesis using chromosome aneuploidy detected by fluorescence in situ hybridization

To visualize the accumulation of chromosome abnormalities in head and neck squamous cell carcinomas (HNSCC) and investigate the extension of the abnormal field, we applied the fluorescence in situ hybridization (FISH) technique to tumor cells and cells collected from a large extension of clinically normal buccal mucosa distant from the tumor in 10 patients. DNA probes specific for 14 human chromosomes (1, 2, 3, 6, 7, 8, 9, 10, 11, 12, 15, 17, X, and Y) were used in dual-target, dual-color FISH assays. Control specimens were collected from oral mucosa of 10 healthy non-smokers, in order to define the tolerance limits for abnormalities, and from 10 healthy smokers. Extensive aneuploidy was detected in most of tumor specimens, more frequently represented by chromosome gains than losses. Interestingly, the clinically normal distant oral regions displayed chromosomal aneuploidies in seven out of the 10 patients tested. These findings support the occurrence of field cancerization in HNSCC. In addition, interphase FISH is demonstrated as an effective technique for detecting chromosome aneuploidy associated with malignancy and a potential tool for non-invasive screening of individuals at high-risk for HNSCC.     

Detection of chromosomal aneuploidy in endometriosis by multi-color fluorescence in situ hybridization (FISH)

Endometriosis affects 10–15% of women of reproductive age and is a common cause of infertility and pelvic pain. Although endometriosis is characterized by abnormal growth or turn-over of cells, the genetic changes involved remain unclear. We employed a multi-color fluorescence in situ hybridization (FISH) strategy to determine the incidence of somatic chromosomal numeric alterations in severe/late stage endometriosis. Using alpha-satellite sequence-specific DNA probes for chromosomes 7, 8, 11, 12, 16, 17, and 18, simultaneous two- and three-color FISH were performed to evaluate the frequency of monosomic, disomic, and trisomic cells in normal control and endometriotic tissue specimens. In one of four endometriosis samples studied, a significantly higher frequency of monosomy for chromosome 17 (14.8%, χ² ₄ = 53.3, P < 0.0001) and 16 (8.8%, χ² ₄ = 11.4, P < 0.05) was observed. An increased number of cells with chromosome 11 trisomy (14.8%, χ² ₄ = 96.2, P < 0.0001) were detected in a second case. In a third case, a distinct colony of nuclei with chromosome 16 monosomy (14.1%, χ² ₄ = 21.39, P < 0.005) was detected. Acquired chromosome-specific aneuploidy may be involved in endometriosis, reflecting clonal expansion of chromosomally abnormal cells. That candidate tumor suppressor genes and oncogenes have been mapped to chromosomes 11, 16, and 17 suggests that chromosomal loss or gain plays a role in the development and/or progression of endometriosis. Received: 27 December 1997 / Accepted: 14 April 1997     

Detection of chromosome 17- and X-bearing human spermatozoa using fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) with DNA probes specific to chromosomes 17 and the X has been applied to human ejaculated sperm. After sperm nuclei were decondensed with EDTA and DTT, biotinylated alpha satellite DNA probes TR17 and TRX were separately used on preparations from thirteen healthy donors. After hybridization 96% of sperm were labelled with the TR17 probe and 48% of sperm were labelled with the TRX probe. Frequencies of 0.33% disomic 17 and 0.29% disomic X sperm were found. The frequencies of diploid sperm were assessed as 0.37% using the TR17 probe and 0.20% using the TRX probe which labelled only one half of the sperm; after correcting the result from the X-probe to 0.40% the two frequencies are very similar.     

Chromatid segregation analysis in native human lymphocyte anaphases using sequential fluorescence in situ hybridization

A sequential multiprobe fluorescence in situ hybridization technique was developed to study the 13, 18, 21, X and Y chromatid segregation in human lymphocytes anaphases cultures without antimitotic treatment. This method was used to know if exist any different chromosomes segregation in lymphocytes from Down syndrome patients and compared it with controls. The results show that the prevalent sequence of centromere separation was X, 13, 21, Y and 18 in Down syndrome patients and Y, 13, X, 21 and 18 in controls. Chromatid segregation in early anaphase was asynchronic for all chromosome pairs studied. Late anaphase showed a frequency of non-disjunction of 4.5% in the controls, affecting only chromosomes 18 and Y; in the Down syndrome patients, the frequency was higher (20.3%) and affected all chromosomes studied. This technique could be applicated to know the incidence of non disjunction in couples with repetitive abortions or in cases with different aneuploidies in the offspring. This revised version was published online in September 2006 with corrections to the Cover Date.     

Multiple fluorescence in situ hybridization

A method for multiple fluorescence in situ hybridization is described allowing the simultaneous detection of more than three target sequences with only three fluorescent dyes (FITC, TRITC, AMCA), respectively emitting in the green, red, and blue. This procedure is based on the labeling of (DNA) probes with more than one hapten and visualisation in multiple colors. The possibility to detect multiple targets simultaneously is important for prenatal diagnosis and the detection of numerical and/or structural chromosome aberrations in tumor diagnosis. It may form the basis for an in situ hybridization based chromosome banding technique.     

Aneuploidy detection in porcine embryos using fluorescence in situ hybridization

In contrast to human embryos, there are very few studies published on the frequency of chromosomal aneuploidy in farm animals. The objectives of this study were to apply a three-color fluorescent in situ hybridization (FISH) method for evaluating aneuploidy in porcine embryos using chromosome-specific DNA probes, establish baseline frequencies of aneuploidy in embryos and compare the results with our previous findings of aneuploidy in spermatozoa and oocytes. The embryos were collected from superovulated gilts, which were slaughtered 48 h after insemination. FISH was performed using probes specific for the centromeric regions of porcine chromosomes 1, 10 and Y. Altogether 403 blastomeres from 114 porcine embryos were successfully investigated. Diploidy was observed in 101 (88.6%) embryos, triploidy in 2 (1.8%) embryos, mosaicism/mixoploidy in 9 (7.9%) embryos, and trisomy for chromosomes 1 or 10 in 2 (1.8%) embryos. No blastomere showed aneuploidy for chromosome Y. These findings correspond with the frequencies of aneuploidy we have found previously in porcine germ cells.     

Advances in fluorescence in situ hybridization

The techniques of in situ hybridization (ISH) are widely applied for analyzing the genetic make-up and RNA expression patterns of individual cells. This review focusses on a number of advances made over the last 5 years in the fluorescence ISH (FISH) field, i.e., Fiber-FISH, Multi-colour chromosome painting, Comparative Genomic Hybridization, Tyramide Signal Amplification and FISH with Polypeptide Nucleic Acid and Padlock probes.     

Total aneuploidy and age-related sex chromosome aneuploidy in cultured lymphocytes of normal men and women

Summary In PHA-cultured lymphocytes, about 8% of metaphases from 32 women were aneuploid compared to 4% of metaphases from 35 men. A significant part of this aneuploidy was characterized by sex chromosome involvement: in women, the loss or gain of X chromosomes; in men, the gain of X chromosomes and the loss or gain of Y chromosomes. The incidence of this aneuploidy was positively age-related for both sexes. Premature division of the X-chromosome centromere was closely associated with X-chromosome aneuploidy in women and men, and appeared to be the mechanism of nondisjunction causing this aneuploidy. Premature centromere division (PCD) indicated a dysfunction of the X-chromosome centromere with aging, and this dysfunction was the basic cause of age-related aneuploidy. A similar mechanism of nondisjunction may operate for the Y chromosome of men, but could not be clearly demonstrated because of the low incidence of Y-chromosome aneuploidy.The balance of the aneuploidy was characterized by chromosome loss and the involvement of all chromosome groups. It was consistent with chromosome loss from metaphase cells damaged during preparation for cytogenetic examination.     

Technique for culturing Macaca mulatta peripheral blood lymphocytes for fluorescence in situ hybridization of whole chromosome paints

The rhesus monkey (Macaca mulatta) has long been an important model in biomedical and behavioral research. The biomedical importance of M. mulatta is due to its 93% genetic similarity with humans and its complex social behavior. The recent sequencing of the M. mulatta genome has enhanced its role in biological research. However, the use of the macaque as an experimental model in cytogenetic assays has been problematic due to difficulties in obtaining large numbers of well-spread cells in metaphase without the use of extremely toxic mitogens such as staphylococcal enterotoxin A (SEA). Here we describe a technique for culturing and producing sufficient numbers of cells in metaphase using the common mitogens phytohemagglutinin (PHA), concanavalin A (ConA), and T-cell growth factor (TCGF) which act synergistically to induce M. mulatta T-lymphocyte division. Using this method we have obtained a mitotic index in 48 h cultures of 12.0+/-2.2 metaphase cells/100 cells (n=5 animals). Fluorescence in situ hybridization with whole chromosome painting of M. mulatta cells was performed with human whole-chromosome probes that labeled the following chromosomes for human (M. mulatta): 1(1), 2q(12), 2p(13), 4(5) pairs in red, and 3(2), 5(6) and 6(4) pairs in green. In humans this probe combination simultaneously paints 3 chromosome pairs in red and 3 in green, whereas in M. mulatta 4 chromosome pairs are labeled in red and 3 pairs are labeled in green. Using this method we show a baseline frequency of 0.026 translocations per 100 whole-genome cell equivalents in peripheral blood lymphocytes obtained from unexposed adolescent non-human primates. This method will add to the usefulness of M. mulatta as an animal model in biomedical research.     

Characterization of Robertsonian translocations by using fluorescence in situ hybridization.

Fluorescence in situ hybridization with five biotin-labeled probes (three alphoid probes, a probe specific for beta-satellite sequences in all acrocentric chromosomes, and an rDNA probe) was used to characterize 30 different Robertsonian translocations, including three t(13;13); one t(15;15), four t(21;21), three t(13;14), two t(13;15), two (13;21), two t(13;22), one t(14;15), eight t(14;21), two t(14;22), and two t(21;22). Of 8 de novo homologous translocations, only one t(13;13) chromosome was interpreted as dicentric, while 19 of 22 nonhomologous Robertsonian translocations were dicentric. The three monocentric nonhomologous translocations included both of the t(13;21) and one t(21;22). Two of 26 translocations studied using the beta-satellite probe showed a positive signal, while rDNA was undetectable in 10 cases studied. These results indicate that most homologous Robertsonian translocations appear monocentric, while the bulk of nonhomologous translocations show two alphoid signals. A majority of the breakpoints localized using this analysis seem to be distal to the centromere and just proximal to the beta-satellite and nuclear-organizing regions.     

Determination of Y chromosome aneuploidy in human sperm nuclei by nonradioactive in situ hybridization.

Sperm nuclei from eight normal, healthy donors were hybridized in situ with the biotin-labeled Y-specific pHY2.1 DNA probe to evaluate the X:Y ratio, the location of the Y chromosome, and the frequency of Y aneuploidy in human sperm. The streptavidine-horseradish-peroxidase and DAB detection system used permitted the unequivocal identification of sperm heads with zero, one, or two hybridization signals and proved superior to either quinacrine staining or radioactive in situ hybridization. The low incidence of 0.27% of sperm nuclei with two Y chromosomes that was found is similar to the frequency of XYY males among newborns. The average proportions of X- and Y-bearing sperm nuclei were 50.3% and 49.4%, respectively, corresponding to the expected 1:1 ratio. The Y heterochromatin was located in the central part of the nucleus in 58% of the Y-carrying sperm cells.     

Detection of ingested bacteria in benthic ciliates using fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) was applied to detect ingested natural bacteria within the food vacuoles of ciliates harvested from the natural sediment. In addition to this important qualitative aspect, FISH was also successfully used to measure the bacterivory of a culture of the ciliate Tetrahymena pyriformis on natural field sediment bacteria. In this feeding experiment, we compared the FISH technique with the only available alternative technique using fluorescently stained sediment (FS-sediment). The ingestion rate of unstained sediment bacteria determined by FISH was 4.6 bacteria per ciliate and hour. In contrast, Tetrahymena pyriformis cells that fed on bacteria from FS-sediment ingested 12.7 bacteria per ciliate and hour. Bacterial abundances in the sediment were equal in both sediment types (4 x 10(8) cells g sediment dry weight(-1)) when determined by DAPI counts. However, when analyzed using DTAF-counts, the number of bacteria in the FS-sediment increased to 9.7 x 10(8) cells g sediment dry weight(-1). From our findings we conclude that bacterivory by ciliates is overestimated when FS-sediment is used because DTAF stains bacteria as well as protein-containing detritus particles, which are also ingested by many ciliates. In contrast, FISH is a direct, a posteriori method that specifically stains phylogenetic lineages, e.g. eubacteria, after ingestion and thereby avoids a false determination of the number of ingested bacteria. Thus this method can also be used for the study of natural ciliate bacterivory in benthic systems.     

Clinical applications of fluorescence in situ hybridization

We review here the application of fluorescence in situ hybridization with chromosome-specific probes to chromosome classification and to detection of changes in chromosome number or structure associated with genetic disease. Information is presented on probe types that are available for disease detection. We discuss the application of these probes to detection of numerical aberrations important for prenatal diagnosis and to detection and characterization of numerical and structural aberrations in metaphase spreads and in interphase nuclei to facilitate tumor diagnosis.     

Routine fluorescence in situ hybridization in soil

The use of fluorescence in situ hybridization (FISH) to identify and enumerate soil bacteria has long been hampered by the autofluorescence of soil particles masking the bacterial signals and because the need of counting hundreds of bacteria in order to achieve statistically reliable data is time consuming. Recently, it was demonstrated that Nycodenz facilitates FISH in soil by concentrating bacteria on membrane filters and avoiding autofluorescent soil particles. We present a routine protocol for FISH in soil including the use of Nycodenz. The protocol allows fast and easy enumeration of hundreds of bacteria. We propose the use of silicon grease coated slides to treat in parallel seven samples per hybridization. Further, we developed a semi-automated approach for the enumeration of bacteria by implementing macros concatenating all steps of the image analyzes in the Image J software. Using Nycodenz, software-assisted bacterial counts statistically matched eye-counts of the same images and it was possible to count 880 DAPI stained bacteria per ten images. Fifty-five percent of these bacteria were co-labelled with the FISH probe specific for the Domain Bacteria, in accordance with recent FISH studies of bacterial populations in bulk soil. With a soil slurry protocol used for comparison, soil particles impaired automatic counts of the bacteria and FISH analysis, and only 88 DAPI stained bacteria per ten images could be counted by eye. With the Nycodenz protocol, 5 mM Na(2)EDTA used as an extractant increased the number of bacteria observed by 49%. In contrast, Tween 20 (1% or 5%) had no significant effect and increased the variability between the samples. Overall, the proposed procedure allows to process a high number of samples and to achieve a time efficient FISH characterization of soil bacterial communities.