The use of non-physiological conditions to isolate fetal cells from maternal blood

Fetal cells are always present in maternal blood starting in the first trimester of pregnancy, however a rapid, simple, and consistent procedure for their isolation for prenatal non-invasive genetic investigation is still lacking. Sensitivity and recovery of fetal cells is jeopardized by the minute amount of circulating fetal cells and their loss during the enrichment procedure. We report here a single-step approach to isolate fetal cells from maternal blood which relies on the use of non-physiological conditions to modify cell densities before their separation in a density gradient and in a newly developed cell separation device. Isolated fetal cells have been investigated using cytochemistry, Soret band absorption microscopy, monoclonal antibodies for epsilon- and gamma-chain-Hb, monoclonal antibody for i-antigen, and by fluorescence in situ hybridization (FISH). Fetal cells were always detected in all 105 maternal blood samples investigated and fetal aneuploidies were correctly diagnosed by FISH, in a pilot study of pathological pregnancies, in fetal cells isolated from maternal blood obtained either before or after invasive procedure.     

Localization of Xho1 repetitive sequences on autosomes in addition to the W chromosome in chickens and its relevance for sex diagnosis

Repetitive sequences of the Xho1 family comprise about 70% of the W chromosome in chickens, are considered to be restricted to this sex chromosome and are used in sex diagnosis. This study investigated the reliability of sex diagnosis on small cell numbers. Fluorescent in situ hybridizations (FISH) with the Xho1 repeat sequence were performed on metaphases and signals were detected on the W chromosome and on some autosomes. Differences in signal intensity suggested that the number of repeats ranged from several thousand on the W chromosome to only a few on the autosomes. These results confirm the report from Clinton (1994) of minor male products in sex specific PCR of the Xho1 repeat. We adopted his PCR protocol to analyze small numbers of cells. FISH on blastomeres and on blood cells from E3 to E11 embryos were compared to PCR results. The FISH procedure as well as the PCR protocol are suitable for sexing. For single cells FISH was slightly more consistent.     

Detection of Acidithiobacillus ferrooxidans in acid mine drainage environments using fluorescent in situ hybridization (FISH)

An important microorganism of acid mine drainage (AMD) and bioleaching environments is Acidithiobacillus ferrooxidans which oxidizes ferrous iron and generates ferric iron, an oxidant. Most investigations to understand microbial aspects of sulfide mineral dissolution have focused on understanding physiological, metabolic, and genetic characteristics of A. ferrooxidans. In this study, a 16S rRNA oligonucleotide probe designated S-S-T.ferr-0584-a-A-18, and labeled at the 5'-end with indocarbocyanine dye (CY3), was used in a fluorescent in situ hybridization (FISH) procedure on pure cultures of nine isolates of A. ferrooxidans. These isolates were recovered from acid mine drainage and mining environments. The probe was also used to detect cells of A. ferrooxidans, recovered from AMD samples, growing on FeTSB and FeSo solid media in a FISH procedure. In addition, the presence of cells of A. ferrooxidans in an environmental water sample from an AMD site in Copper Cliff, Ontario, Canada was analyzed using the FISH technique. Probe specificity was first confirmed with A. ferrooxidans ATCC 19859 (positive control) and Acidithiobacillus thiooxidans ATCC 19377, Acidiphilium acidophilum ATCC 27807, and Lactobacillus plantarum ATCC 8014 (negative controls). Positive and negative control cells were also used to determine optimal stringency conditions for hybridizations with the probe. Cells of the nine isolates of A. ferrooxidans stained positive, although the fluorescent signal varied in intensity from isolate to isolate. Colonies of A. ferrooxidans from the environmental water sample of the AMD site were recovered only on FeTSB solid medium after 22 days of incubation. The probe was able to detect cells of A. ferrooxidans in a FISH procedure. However, no cells of A. ferrooxidans were detected in the AMD water sample without cultivation. Thus, probe S-S-T.ferr-0584-a-A-18 hybridized effectively with cells of A. ferrooxidans recovered from pure cultures but failed to directly detect cells of A. ferrooxidans in the AMD site.     

In situ analysis of native microbial communities in complex samples with high particulate loads

In the present study a procedure combining a cell extraction method and Fluorescence In Situ Hybridization (FISH) for molecular monitoring and quantification of bacteria in soil and aquifer samples is presented. FISH was applied to bacterial cells extracted from the matrix by density gradient centrifugation. This separation method was applied to soil and aquifer samples and produced high cell recovery of 76.5%+/-4.4 and 78.0%+/-3.2, respectively. FISH, performed on the harvested cells, permitted a perfect visualization and quantification of bacteria. This approach is therefore promising for in situ detection of indigenous bacterial communities in complex samples.     

A combination of direct viable count and fluorescence in situ hybridization for specific enumeration of viable Lactobacillus delbrueckii subsp.bulgaricus and Streptococcus thermophilus

Aims: We have developed a direct viable count (DVC)‐FISH procedure for quickly and easily discriminating between viable and nonviable cells of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains, the traditional yogurt bacteria. Methods and Results: direct viable count method has been modified and adapted for Lact. delbrueckii subsp. bulgaricus and Strep. thermophilus analysis by testing different times of incubation and concentrations of DNA‐gyrase inhibitors. DVC procedure has been combined with fluorescent in situ hybridization (FISH) for the specific detection of viable cells of both bacteria with specific rRNA oligonucleotide probes (DVC‐FISH). Of the four antibiotics tested (novobiocin, nalidixic acid, pipemidic acid and ciprofloxacin), novobiocin was the most effective for DVC method and the optimum incubation time was 7 h for both bacteria. The number of viable cells was obtained by the enumeration of specific hybridized cells that were elongated at least twice their original length for Lactobacillus and twice their original size for Streptococcus. Conclusions: This technique was successfully applied to detect viable cells in inoculated faeces. Significance and Impact of the Study: Results showed that this DVC‐FISH procedure is a quick and culture‐independent useful method to specifically detect viable Lact. delbrueckii subsp. bulgaricus and Strep. thermophilus in different samples, being applied for the first time to lactic acid bacteria.     

Detection of cell-cycle stage by fluorescence in situ hybridization: its application in human interphase cytogenetics.

Distinct cell-cycle-dependent changes in the conformation of centromeric chromatin in a specific human chromosome containing alpha-satellite DNA have been demonstrated by fluorescence in situ hybridization (FISH). This method, based upon specific FISH signal morphology, allows simultaneous analysis of chromosomal aneuploidy and detection of specific cell-cycle stage(s) of human tumor and/or normal cell populations in a single preparation of interphase cells. This interphase cytogenetic procedure might prove useful for both basic and clinical research involving human cells.     

Seven-hour fluorescence in situ hybridization technique for enumeration of Enterobacteriaceae in food and environmental water sample

AIMS: A fluorescent in situ hybridization (FISH) technique using an Enterobacteriaceae-specific probe (probe D) to target 16S rRNA was improved in order to enumerate, within a single working day, Enterobacteriaceae present in food and environmental water samples. METHODS AND RESULTS: In order to minimize the time required for the FISH procedure, each step of FISH with probe D was re-evaluated using cultured Escherichia coli. Five minutes of ethanol treatment for cell fixation and hybridization were sufficient to visualize cultured E. coli, and FISH could be performed within 1 h. Because of the difficulties in detecting low levels of bacterial cells by FISH without cultivation, a FISH technique for detecting microcolonies on membrane filters was investigated to improve the bacterial detection limit. FISH with probe D following 6 h of cultivation to grow microcolonies on a 13 mm diameter membrane filter was performed, and whole Enterobacteriaceae microcolonies on the filter were then detected and enumerated by manual epifluorescence microscopic scanning at magnification of x100 in ca 5 min. The total time for FISH with probe D following cultivation (FISHFC) was reduced to within 7 h. FISHFC can be applied to enumerate cultivable Enterobacteriaceae in food (above 100 cells g-1) and environmental water samples (above 1 cell ml-1). CONCLUSIONS: Cultivable Enterobacteriaceae in food and water samples were enumerated accurately within 7 h using the FISHFC method. SIGNIFICANCE AND IMPACT OF THE STUDY: A FISHFC method capable of evaluating Enterobacteriaceae contamination in food and environmental water within a single working day was developed.     

Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation.

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30 kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.     

Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30 kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.     

Analysis of chromosomal equipment in spermatozoa of a 46,XY/47,XY/+8 male by means of multicolour fluorescent in situ hybridization: confirmation of a mosaicism and evaluation of risk for offspring

Trisomy 8 is generally associated with chromosomal mosaicism and occurs de novo, with relatively well-defined clinical manifestations, ranging from minimal effects to severe malformations. Mosaicism is often difficult to ascertain and the confirmation of diagnosis requires several chromosomal investigations on a variety of tissues. We present a case of mosaic trisomy 8 fortuitously found in a healthy 30-year-old man during a cytogenetic investigation for several spontaneous abortions: 8% of the lymphocyte metaphases showed a 47,XY,+8 karyotype. Fluorescent in situ hybridization (FISH) with the probes pJM.128 and L1.84 was performed on decondensed interphase spermatozoa. Of the 25 000 analysed cells, 398 spermatozoa (1.59%) exhibited a hybridization pattern compatible with a chromosome 8 disomy; the frequency was higher than in either sperm control populations (0.17% and 0.21%). This result is in agreement with the existence of trisomy 8 mosaicism in the gonads and germ cells. FISH on decondensed interphase spermatozoa spreads is thus an easy non-invasive procedure that can be used to complement mosaicism diagnosis in tissue other than blood. Moreover, FISH provides interesting data for characterizing the risk for offspring. Received: 11 July 1996 / Revised: 26 August 1996     

Combination of lamin immunocytochemistry and in situ hybridization for the analysis of chromosome copy numbers in tumor cell areas with high nuclear density

We describe the application of lamin immunocytochemistry (ICC) and single- or double-target fluorescence in situ hybridization (FISH) on 4 microm thick frozen tissue sections as a method to facilitate scoring of aberrant chromosome copy numbers in colonic tumors. Analysis of FISH signals in colon tissue sections is often hampered by overlap and truncation of epithelial nuclei, due to the density of the epithelial cells. Furthermore, on the basis of nuclear staining it is often difficult to determine whether or not nuclei are overlapping, or adjoining. Therefore, reliable evaluation of (F)ISH signals to screen for genomic changes was until now mainly restricted to isolated nuclei obtained from relatively thick tissue sections. In this study the applicability of lamin ICC, to stain the nuclear periphery and to distinguish individual nuclei, combined with the FISH procedure is explored to solve this problem for colon epithelium. For ICC we applied the alkaline phosphatase (APase)-Fast Red detection method, since the fluorescent precipitate of this reaction resists extensive proteolytic digestion as needed for efficient FISH on tissue sections. Chromosome copy numbers could easily be determined in 4 microm thick frozen tissue sections by combining lamin ICC and FISH. The ratio of the copy numbers of the chromosomes 7 and 17 could be determined in frozen tissue sections after combined lamin ICC and double-target FISH. It is concluded that the combination of lamin ICC and FISH improves chromosome copy number analysis and can be used to investigate genomic changes in different tumor compartments in thin frozen tissue sections.     

Diagnosis of bovine freemartinism by fluorescence in situ hybridization on interphase nuclei using a bovine Y chromosome-specific DNA probe

A heifer co-twin to a bull, in most cases, is a sterile freemartin which needs to be identified and culled from replacement stock. Various methods are available for the diagnosis of freemartinism, but none is ideal in terms of speed, sensitivity, or specificity. The present study was thus conducted to develop and validate a satisfactory fluorescence in situ hybridization procedure on interphase nuclei (I-FISH) for identifying the bovine XX/XY-karyotypic chimerism, the hallmark of freemartinism. A 190-bp DNA FISH probe containing the bovine male-specific BC1.2 DNA sequence was synthesized and labeled with digoxigenin by PCR. The FISH was performed on metaphase spreads and interphase nuclei of blood lymphocytes. Upon FISH, the probe expectedly bound to the nucleus of the male cell or to a region of the p12 locus of the Y chromosome. Twenty-four young heterosexual twins (Holstein-Friesian and Korean Cattle breeds; 10 pairs and 4 singletons) were analyzed in the present study; all but three exhibited the XX/XY-karyotypic chimerism to varying extents in both I-FISH and karyotyping. One heifer was identified to have 100% XX cells by both analyses, whereas two bulls were judged as 100% XY- and XX/XY-chimeric karyotypes by karyotyping and I-FISH, respectively. Nevertheless, the ratios of the XY to XX cells in these animals were very similar between the two analyses. In conclusion, the present I-FISH was a rapid and reliable procedure that can be used for early-life diagnosis of bovine freemartinism.     

Regulation of c-myc through intranuclear localization of its RNA subspecies

We used fluorescence in situ hybridization (FISH) to detect c-myc RNA subspecies in human COLO 320DM tumor cells. Although the FISH procedure removed the majority of RNAs from the nucleolus, c-myc RNA continued to be detected in both the nucleoplasm and nucleolus. This finding suggests stable association between c-myc RNA and the nucleolus. Nucleolar accumulation of c-myc RNA appeared to be temporally regulated by cell-cycle progression. Hybridization with exon- and strand-specific RNA probes indicated that the non-protein coding exon 1 plays a novel role in determining the subnuclear localization of c-myc RNA. Antisense RNA targeting exon 2 localized only with nucleoplasmic foci, where it might interact with the sense strand. Thus, c-myc gene expression may be regulated by intranuclear localization of its RNA.     

Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30?kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.     

Development of a fluorescence in situ hybridization method for cheese using a 16S rRNA probe

A 16S rRNA fluorescence in situ hybridization (FISH) method for cheese was developed to allow detection in situ of microorganisms within the dairy matrix. An embedding procedure using a plastic resin was applied to Stilton cheese, providing intact embedded cheese sections withstanding the hybridization reaction. The use of a fluorescein-labelled 16S rRNA Domain Bacteria probe allowed observation of large colonies of microbial cells homogeneously distributed in the cheese matrix. FISH experiments performed on cheese suspensions provided images of the different microbial morphotypes occurring. The technique has great potential to study the spatial distribution of microbial populations in situ in foods, especially where the matrix is too fragile to allow manipulation of cryosections.     

Collecting human spermatozoa onto filters for FISH. Application to the study of extreme oligozoospermia

OBJECTIVE: To describe a simple method of fluorescence in situ hybridization (FISH) performed on germ cells collected on filters that allows the study of aneuploid sperm frequency in cases of extreme oligozoospermia. STUDY DESIGN: The aneuploidy rates for chromosomes X, Y and 18 were analyzed in three groups to test the reliability of the filtration-FISH procedure when compared to controls and to evaluate the frequency of aneuploid spermatozoa in five oligospermic men. RESULTS: Filtration of semen samples before FISH does not modify the basal rate of aneuploidy even after various sperm dilutions. In oligospermic men, the frequency of gonosomal aneuploidies is significantly increased. CONCLUSION: The filtration-FISH technique is a powerful method of analyzing chromosomal abnormalities in sperm nuclei in men with extreme oligozoospermia.     

Fluorescence in situ hybridization on paraffin-embedded abortion material as a means of retrospective chromosome analysis

A fluorescence in situ hybridization (FISH) procedure was used to detect chromosome abnormalities in archival abortion material. Nuclei were isolated from 50-m-thick tissue blocks from 18 selected and karyotyped abortions. Five probes for repetitive centromeric sequences of chromosomes 1, 16, 18, X and Y were used. For each chromosome, at least 200 nuclei were scored blindly, i.e. without knowledge of the karyotype. The FISH results obtained were compatible with the cytogenetic data in 14 cases. There were four discrepancies. Two of these were observed for cases karyotyped as trisomy 16. Furthermore, FISH results showed trisomy 18 in two cases having normal chromosomes 18 and 18q+, respectively. The latter case was not discrepant if the structural rearrangement involved chromosome 18 material. The remaining discrepancies could be explained by chromosomal mosaicism. Admixture of normal maternal cells was also noted. It is concluded that FISH can be used to study retrospectively the presence of chromosome abnormalities in abortion material. However, the quality obtained after the use of fresh material is superior.     

Simultaneous visualization of rDNA clusters and the nucleolus by combining fluorescence in situ hybridization and silver staining.

We designed two procedures to visualize simultaneously clusters of ribosomal RNA genes (rDNA) and the nucleolus in plant cells. The procedures combine fluorescence in situ hybridization (FISH) to visualize the rDNA clusters and silver staining to observe the nucleolus. When FISH is followed by silver staining, many minute FISH signals are localized in the nucleolus, and several large FISH signals are seen on the nucleolar periphery. When FISH was applied to the specimens with silver nitrate staining, large FISH signals were visualized in the nucleoplasm associated with the nucleolar periphery, but no signals were seen in the nucleoli. Thus, the two combinations of FISH and silver staining provided different details regarding the arrangement of rDNA clusters in the nucleolus of plant cells.     

A digital imaging procedure for seven-probe-labeling FISH (Rainbow-FISH) and its application to estuarine microbial communities

For multi-probe-labeling fluorescence in situ hybridization (FISH), a digital imaging procedure was developed consisting of systematic background noise reduction and target signal equalization using a hue, saturation, value color partitioning technique. By the combined application of seven DNA probes, each labeled with three fluorochromes at maximum, seven kinds of cultured type strains were distinguished in a microscopic field simultaneously. Using this seven-probe-labeling FISH (Rainbow-FISH), several phylogenetic groups of microbes that occur frequently in aquatic environments, such as Alpha-, Beta- and Gammaproteobacteria, Cytophaga-Flavobacterium and Actinobacteria, were identified and quantified. The total counts of cells specified by Rainbow-FISH were in the range of 96-108% of those of general FISH, showing that the method is highly reliable for quantitative population analysis. Analyzing samples obtained at points along a river to a sea, we found a reverse population change in two groups: apparent decreases in Betaproteobacteria but gradual increases in Gammaproteobacteria. This method provides a platform toward the improvement of semiautomatic analysis of aquatic microbes under various metabolic conditions.