Avidin-EITC: an alternative to avidin-FITC in confocal scanning laser microscopy.

Detection of fluorescein-5-isothiocyanate (FITC)-labeled conjugates is suboptimal in two-color confocal scanning laser microscopy (CLSM). This limits the detection of small, dimly fluorescent targets. We explored the possible advantages of applying eosin-5-isothiocyanate (EITC) conjugated to avidin (Av-EITC) as an alternative for Av-FITC in CSLM. Despite the lower quantum efficiency of EITC, we found that the measured Av-EITC and Av-FITC emission intensities were similar as a result of the standard filter combinations used for simultaneous two-color detection in the Bio-Rad MRC 600 CSLM. The advantage of Av-EITC was that its fading characteristics compared very favorably to those of Av-FITC. An excitation intensity-dependent increase in Av-EITC fluorescence was observed, followed by an exponential decrease. This increase in fluorescence allows longer observation times, averaging of several scans without loss of brightness, and thus detection of dimly fluorescent targets by CSLM.

Effect of bone decalcification procedures on DNA in situ hybridization and comparative genomic hybridization. EDTA is highly preferable to a routinely used acid decalcifier.

Decalcification is routinely performed for histological studies of bone-containing tissue. Although DNA in situ hybridization (ISH) and comparative genomic hybridization (CGH) have been successfully employed on archival material, little has been reported on the use of these techniques on archival decalcified bony material. In this study we compared the effects of two commonly used decalcifiers, i.e. , one proprietary, acid-based agent (RDO) and one chelating agent (EDTA), in relation to subsequent DNA ISH and CGH to bony tissues (two normal vertebrae, six prostate tumor bone metastases with one sample decalcified by both EDTA and RDO). We found that RDO-decalcified tissue was not suited for DNA ISH in tissue sections with centromere-specific probes, whereas we were able to adequately determine the chromosomal status of EDTA-decalcified material of both control and tumor material. Gel electrophoresis revealed that no DNA could be successfully retrieved from RDO-treated material. Moreover, in contrast to RDO-decalcified tumor material, we detected several chromosomal imbalances in the EDTA-decalcified tumor tissue by CGH analysis. Furthermore, it was possible to determine the DNA ploidy status of EDTA- but not of RDO-decalcified material by DNA flow cytometry. Decalcification of bony samples by EDTA is highly recommended for application in DNA ISH and CGH techniques.

DNA in situ hybridization (interphase cytogenetics) versus comparative genomic hybridization (CGH) in human cancer: detection of numerical and structural chromosome aberrations.

DNA in situ hybridization techniques for cytogenetic analyses of human solid cancers are nowadays widely used for diagnostic and research purposes. The advantage of this methodology is that it can be applied to cells in the interphase state, thereby circumventing the need for high-quality metaphase preparations for karyotypic evaluation. In situ hybridization (ISH) with chromosome specific (peri)centromeric DNA probes, also termed "interphase cytogenetics", can be used to detect numerical changes, whereas comparative genomic hybridization (CGH) discloses chromosomal gains and losses, i.e. amplifications and deletions. We wanted to compare both methods in human solid tumors, and for this goal we evaluated ISH and CGH within a set of 20 selected prostatic adenocarcinomas. Chromosomes 7 and 8 were chosen for this analysis, since these chromosomes are frequently altered in prostate cancer. ISH with chromosome 7 and 8 specific centromeric DNA probes was applied to standard, formalin-fixed and paraffin-embedded, histological sections for numerical chromosome analysis. CGH with DNA's, extracted from the same histologic area of the archival specimens, was used for screening of gains and losses of 7 and 8. ISH with centromeric probes distinguished a total of 26 numerical aberrations of chromosome 7 and/or 8 in the set of 20 neoplasms. In the same set CGH revealed a total of 35 losses and gains. CGH alterations of 7 and 8 were seen in twenty-two of the 26 chromosomes (85%) that showed aberrations in the ISH analysis. Concordance between ISH and CGH was seen in 11 (of 26; 42%) chromosomes. Eight chromosomes were involved in gains (5 x #7, 3 x #8), three in losses (3 x #8). This included both complete (3/11) and partial (8/11) CGH confirmation of the numerical alteration. Partial CGH confirmation was defined as loss or gain of a chromosome arm with involvement of the centromeric region. In the majority of these cases it concerned a whole chromosome arm, mostly the long arm. We conclude that generally a fair correlation was found between ISH and CGH in interphase preparations of a series of prostate cancers. However, when specified in detail, most of the numerical ISH aberrations were only partly represented in the CGH analysis. On the one hand, it suggests that CGH does not adequately discriminate numerical abnormalities. On the other hand, it likely implies that not all numerical changes, as detected by interphase cytogenetics, are truly involving the whole chromosome. A part of these discrepancies might be caused by structural mechanisms, most notably isochromosome formation.

Gain of FAM123B and ARHGEF9 in an Obese Man with Intellectual Disability, Congenital Heart Defects and Multiple Supernumerary Ring Chromosomes.

In a 24-year-old man with mild intellectual disability, congenital heart defects and obesity, we identified up to 4 small supernumerary marker chromosomes (sSMCs) in blood metaphases. The ring-shaped sSMCs were derived from chromosomes 11, 12 and X as well as a fourth, unidentified chromosome. In interphase nuclei of epithelial cells from the urinary tract and buccal mucosa, the presence of the r(11), r(12) and r(X) was confirmed by FISH. Using Illumina Infinium 317K SNP-arrays, we detected 3 copies of the pericentromeric regions of chromosomes 11, 12 and X. The r(X) was present in 84-89% of cells in the various tissues examined, lacks the XIST gene, but contains FAM123B, a potential dosage-sensitive candidate gene for congenital cardiac abnormalities, and ARHGEF9, a candidate gene for intellectual disability. ARHGEF9 encodes collybistin (CB), which is required for localization of the inhibitory receptor-anchoring protein gephyrin and for formation and maintenance of postsynaptic GABAA and glycine receptors. We propose that the 2-fold increase in dosage of ARHGEF9 disturbs the stoichiometry of CB with its interacting proteins at inhibitory postsynapses. SNP alleles and short tandem repeat markers on the r(11) and r(X) were compatible with a maternal origin of both sSMCs through a meiosis II error. The sSMCs may have resulted from predivision chromatid nondisjunction, leading to anaphase lagging, followed by incomplete degradation of the supernumerary chromosomes.

Numerical chromosomal abnormalities in equine embryos produced in vivo and in vitro.

Chromosomal aberrations are often listed as a significant cause of early embryonic death in the mare, despite the absence of any concrete evidence for their involvement. The current study aimed to validate fluorescent in situ hybridization (FISH) probes to label specific equine chromosomes (ECA2 and ECA4) in interphase nuclei and thereby determine whether numerical chromosome abnormalities occur in horse embryos produced either in vivo (n = 22) or in vitro (IVP: n = 20). Overall, 75% of 36,720 and 88% of 2,978 nuclei in the in vivo developed and IVP embryos were analyzable. Using a scoring system in which extra FISH signals were taken to indicate increases in ploidy and "missing" signals were assumed to be "false negatives," 98% of the cells were scored as diploid and the majority of embryos (30/42: 71%) were classified as exclusively diploid. However, one IVP embryo was recorded as entirely triploid and a further seven IVP and four in vivo embryos were classified as mosaics containing diploid and polyploid cells, such that the incidence of apparently mixoploid embryos tended to be higher for IVP than in vivo embryos (P = 0.118). When the number of FISH signals per nucleus was examined in more detail for 11 of the embryos, the classification as diploid or polyploid was largely supported because 2,174 of 2,274 nuclei (95.6%) contained equal numbers of signals for the two chromosomes. However, the remaining 100 cells (4.4%) had an uneven number of chromosomes and, while it is probable that many were artefacts of the FISH procedure, it is also likely that a proportion were the result of other types of aneuploidy (e.g., trisomy, monosomy, or nullisomy). These results demonstrate that chromosomally abnormal cells are present in morphologically normal equine conceptuses and suggest that IVP may increase their likelihood. Definitive distinction between polyploidy, aneuploidy and FISH artefacts would require the use of more than one probe per chromosome and/or probes for more than two chromosomes.

Interphase cytogenetics and comparative genomic hybridization of human epithelial cancers and precursor lesions.

The accuracy of cytogenetic analyses of human solid cancers has improved enormously over the past decade by the introduction and refinement of DNA in situ hybridization (ISH) techniques. This methodology can be applied to cells in the interphase state, thereby making it an excellent tool for the delineation of chromosomal aberrations in solid tumors. The use of non-isotopic ISH to intact and disaggregated cancer specimens will be discussed, as well as comparative genomic hybridization (CGH) with tumor-derived DNAs. In this review we will focus on hybridocytochemical interphase approaches for the detection of chromosomal changes in frequently occurring human epithelial malignancies, e.g., breast, lung, and prostate carcinomas. We will further discuss the use of ISH procedures for the genetic analysis of precursor conditions leading to invasive carcinomas. Knowledge concerning these precancerous conditions is increasing, and its importance in cancer prevention has been recognized. Interphase cytogenetics by ISH, as well as CGH, with DNAs derived from microdissected, precancerous, dysplastic tissue areas will increase our understanding of these lesions, both at the investigative and diagnostic levels.

The nuclear position of pericentromeric DNA of chromosome 11 appears to be random in G0 and non-random in G1 human lymphocytes.

The nuclear topography of pericentromeric DNA of chromosome 11 was analyzed in G0 (nonstimulated) and G1 [phytohemagglutinin (PHA) stimulated] human lymphocytes by confocal microscopy. In addition to the nuclear center, the centrosome was used as a second point of reference in the three-dimensional (3D) analysis. Pericentromeric DNA of chromosome 11 and the centrosome were labeled using a combination of fluorescent in situ hybridization (FISH) and immunofluorescence. To preserve the 3D morphology of the cells, these techniques were performed on whole cells in suspension. Three-dimensional images of the cells were analyzed with a recently developed 3D software program (Interactive Measurement of Axes and Positioning in 3 Dimensions). The distribution of the chromosome 11 centromeres appeared to be random during the G0 stage but clearly non-random during the G1 stage, when the nuclear center was used as a reference point. Further statistical analysis of the G1 cells revealed that the centromeres were randomly distributed in a shell underlying the nuclear membrane. A topographical relationship between the centrosome and the centromeres appeared to be absent during the G0 and G1 stages of the cell cycle.

Comparison of automated and manual analysis of interphase in situ hybridization signals in tissue sections and nuclear suspensions.

In this study we compared visual and automated analyses of interphase in situ hybridization (ISH) signals in five prostatic tumor specimens and one normal prostate sample, both in tissue sections and nuclear suspensions. The advantage of tissue sections is preservation of tissue morphology allowing precise analysis of tumor cells only. The advantage of nuclear suspensions is easier access to automated analysis, due to their disaggregated and dispersed cellular appearance. The samples were hybridized with probes for the (peri)centromeric regions of chromosome 1 and Y. The number of ISH signals per nucleus was counted both manually and automatically by means of a commercially available image analysis system. After image analysis the results were interactively corrected using a gallery display. The automatic and manual counts, before and after interactive correction, were then statistically evaluated. We found no significant differences in overall distributions between the automated and the manual counts, before as well as after correction. This was observed for both tissue sections and cellular suspensions. It is therefore concluded that automated analysis of ISH signals is feasible in both nuclear suspensions and in tissue sections, despite a low percentage of nuclei that could be measured on the latter.

Universal linkage system: an improved method for labeling archival DNA for comparative genomic hybridization.

Comparative genomic hybridization (CGH) has become a powerful technique for studying gains and losses of DNA sequences in solid tumors. Importantly, DNA derived from archival tumor tissue is also applicable in CGH analysis. However, DNA isolated from routinely processed, formalin-fixed, paraffin-embedded tissue is often degraded, with the bulk of DNA showing fragment sizes of only 400-750 bp. Enzymatic labeling of archival DNA by standard nick translation (NT) decreases DNA size even further, until it becomes too small for CGH (<300 bp). This study presents application in CGH of a commercially available, non-enzymatic labeling method, called Universal Linkage System (ULS), that leaves the DNA fragment size intact. To compare the effect of chemical labeling of archival DNA by ULS vs. enzymatic by NT on the quality of CGH, DNA derived from 16 tumors was labeled by both ULS and NT. In those cases (n = 8), in which the bulk of DNA had a fragment size of 400-1,000 bp, CGH was successful with ULS-labeled probes, but not with NT-labeled probes. In the DNA samples (n = 6) with a fragment size > 1 kb, the intensity of CGH signals was comparable for both ULS- and NT-labeled probes, but CGH with ULS-labeled samples showed a high, speckled, background, which seriously hampered image analysis. In the remaining two cases, which had evenly distributed DNA fragment sizes (range 250-5,000 bp), CGH was successful with both labeling methods. Using DNA fragment size < 1 kb as a selection criterion for ULS labeling, we were able to obtain good quality CGH of a large panel (n = 77) of a variety of archival solid tumors. We conclude that ULS is an excellent labeling method for performing CGH on small-fragment-sized DNA.

Interphase in situ hybridization to disaggregated and intact tissue specimens of prostatic adenocarcinomas.

A comparative study was performed of interphase in situ hybridization (ISH) to deparaffinized 4-microns tissue sections and nuclear suspensions from eight prostatic adenocarcinomas, as well as one normal prostatic control. Whole nuclear suspensions were derived from the same tumor areas to evaluate differences of ISH to truncated versus whole nuclei. DNA probes specific for the centromeres of chromosome 1, 7, 8, 10, and Y were used for detection of numerical chromosomal changes and aneuploidy. In six adenocarcinomas chromosome aberrations (+7, +8, -8, -10, -Y) were seen. However, ISH to sections revealed focal aberrations (-10, -Y) in four cases that could not be distinguished in the suspensions. Chromosomal alterations occurring in larger tumor areas were also detected in the nuclear suspensions. Chromosome copy number changes, especially gains, were better discriminated in the nuclear suspensions. The rate of ISH aneuploidy seen in nuclear suspensions corresponded with that observed in the tissue sections (P < 0.01). Ploidy patterns as assessed by ISH to sections and nuclear suspensions were in concordance with DNA flow cytometry (both P < 0.001). We conclude that both section and suspension ISH were able to accurately detect aneuploidy and numerical chromosomal aberrations occurring in larger histological areas. However, section ISH was also capable of revealing (small) focal cytogenetic abnormalities, due to a precise analysis of only target cells. Focal abnormalities were not detected by suspension ISH, probably due to an admixture of non-aberrant tumor cells and stromal elements.