Detection of trisomy 12 by fluorescence in situ hybridization on archival cytopathologic material in chronic lymphocytic leukemia/small lymphocytic lymphoma

OBJECTIVE: To evaluate fluorescence in situ hybridization (FISH) for the detection of trisomy 12 in archival cytologic specimens of chronic lymphocytic leukemia/small lymphocytic lymphoma. STUDY DESIGN: The cytopathology database was searched for all cases of chronic lymphocytic leukemia/small lymphocytic lymphoma. Six cases of chronic lymphocytic leukemia/small lymphocytic lymphoma obtained by fine needle aspiration and one case of small lymphocytic lymphoma with plasmacytoid features were analyzed for trisomy 12 by FISH. These cases had been archived between 1 week to 16 months prior to analysis. RESULTS: We detected trisomy 12 in four of the six cases of small lymphocytic lymphoma/chronic lymphocytic leukemia. The case of small lymphocytic lymphoma with plasmacytoid features was negative for trisomy 12. CONCLUSION: Detection of trisomy 12 by FISH can be effectively performed on routinely prepared, stained and coverslipped archival cytologic material.     

Detection of viral DNA and RNA by in situ hybridization

Using cloned restriction endonuclease fragments of Herpes simplex virus (HSV), human papillomavirus (HPV), and cytomegalovirus (CMV) DNA as probes, viral DNA and RNA sequences have been detected in human tissues. The probes were labeled either with a radioactive isotope, for subsequent detection by autoradiography, or with biotin. This latter technique has been successfully used to visualize HPV DNA in tissues that have been fixed in formalin and embedded in paraffin, and is therefore of value in retrospective studies of histological specimens. HPV DNA was detected under non-stringent conditions (Tm = -42 degrees C) with heterologous probes in plantar and common warts, laryngeal papillomas, and anogenital condylomas. The specific type of HPV was established using stringent hybridization conditions (Tm = - 17 degrees C). Results from these and from malignant tissues show the distribution and localization of HSV and HPV RNA and DNA sequences in malignancies of squamous cell origin in the anogenital region. Both HSV and HPV DNA sequences have occasionally been detected in the same tumor, providing a further impetus to test the hypothesis that an initiator-promoter relationship might involve these common human viruses in the development of some tumors.     

Detection and differentiation of chlamydiae by fluorescence in situ hybridization

Chlamydiae are important pathogens of humans and animals but diagnosis of chlamydial infections is still hampered by inadequate detection methods. Fluorescence in situ hybridization (FISH) using rRNA-targeted oligonucleotide probes is widely used for the investigation of uncultured bacteria in complex microbial communities and has recently also been shown to be a valuable tool for the rapid detection of various bacterial pathogens in clinical specimens. Here we report on the development and evaluation of a hierarchic probe set for the specific detection and differentiation of chlamydiae, particularly C. pneumoniae, C. trachomatis, C. psittaci, and the recently described chlamydia-like bacteria comprising the novel genera Neochlamydia and PARACHLAMYDIA: The specificity of the nine newly developed probes was successfully demonstrated by in situ hybridization of experimentally infected amoebae and HeLa 229 cells, including HeLa 229 cells coinfected with C. pneumoniae and C. trachomatis. FISH reliably stained chlamydial inclusions as early as 12 h postinfection. The sensitivity of FISH was further confirmed by combination with direct fluorescence antibody staining. In contrast to previously established detection methods for chlamydiae, FISH was not susceptible to false-positive results and allows the detection of all recognized chlamydiae in one single step.     

Detection of legumin gene DNA sequences in pea by in situ hybridization

In order to detect low copy number sequences in pea using biotin-labelled probes we optimised some aspects of the in situ hybridization technique. We found protoplast preparations to be superior to standard squashes in terms of their signal: noise ratio. Heat and alkali denaturation of chromosomal DNA were both more effective than acid denaturation. A comparison of antibody-fluorochrome and streptavidin-enzyme conjugates showed the streptavidin-alkaline phosphatase conjugate to be the most sensitive detection system. Using the optimised method, we were able to detect a single site for a 13.5 kb legumin gene clone.     

Detection of single-copy genes by nonisotopic in situ hybridization on human chromosomes

Summary A technique of in situ hybridization on metaphase chromosomes with biotinylated DNA probes is described. This technique was used to localize unique DNA sequences on chromosomes and allowed a localization of two probes 1.8 and 1.3 kb long. The hybridization signal appears like two, twin, spots on the two sister chromatids, allowing a clear distinction from the background. Moreover a chromosomal localization is possible by counting a relatively small number of mitoses compared with the technique using ³H-labeled DNA probes.     

Detection and quantification of rRNA by high-resolutionin situ hybridization in pollen grains

We examined changes in the localization of cytoplasmic rRNA during pollen development inNicotiana tabacum SR-1. The rRNA was visualized byin situ hybridization, and the signal intensity of rRNA in microspore, vegetative and generative cell was quantified by microphotometry. The amount of rRNA per microspore or pollen section increased about 5 times from microspore to mature pollen grain and kept increasing even in the late stage of pollen development after PMI. The increase of rRNA occur in both vegetative and generative cells. The results suggest that synthesis of rRNA occur even after PM I in both vegetative and generative cells.     

Detection of the oyster parasite Bonamia ostreae by fluorescent in situ hybridization

Bonamia ostreae is an economically significant protistan parasite of the flat oyster Ostrea edulis in Europe and North America. Management of this parasite depends partly upon its reliable identification in wild and aquacultured oyster populations, but B. ostreae is small and difficult to detect by traditional microscopic methods. We designed a fluorescent in situ hybridization (FISH) assay to sensitively detect B. ostreae in standard histopathological sections of B. ostreae-infected oysters using fluorescently labeled DNA oligonucleotide probes. Hybridization using a cocktail of 3 presumptively B. ostreae-specific, fluorescein iso(thio)cyanate (FITC)-labeled oligonucleotides produced an unambiguous staining pattern of small green rings inside infected oyster hemocytes that was easily distinguished from host tissue background. This pattern is diagnostic for B. ostreae. A negative control cocktail of oligonucleotides containing 2 mismatches relative to target sequences, on the other hand, failed to hybridize at all. B. ostreae-specific probes did not cross-react with a related protist, Haplosporidium nelsoni.     

Detection of a single-copy gene on plant chromosomes by in situ hybridization

Summary DNA recombinant technology, combined with improvements in hybridization efficiency and quality of chromosome spreads, has made the method of in situ hybridization a reliable tool for gene mapping used by mammalian cytogeneticists to complement other methods. By appropriate alterations of the method, we demonstrate that detection of unique genes can be achieved along plant chromosomes despite some inherent disadvantages of the plant material. Using genomic subclones homologous to 6.6 kb of the single-copy chalcone synthase gene in parsley, we report the first example of chromosomal detection and localization of a unique endogenous gene in plants.     

Trisomy 8p: unusual origin detected by fluorescence in situ hybridization

Summary Chromosomal analysis of a neonate with brain and heart abnormalities revealed trisomy for 8p. The mother's karyotype showed 47 chromosomes with one chromosome 8 being represented as two separate chromosomes, an acrocentric 8p and a telocentric 8q. Gbanding and silver staining revealed a satellite and nucleolus organizing region (NOR) on the 8p. Centromericspecific probes to the centromeres of chromosomes 8, 15, 13/21, 22 and the acrocentric chromosomes revealed that only the 8q centromere was of chromosome-8 origin, while the 8p centromere was of chromosome-14 origin.     

Detection of DNA sequences in Plasmodium berghei by means of in situ hybridization

A non-radioactive in situ hybridization technique, used to map unique DNA sequences to plant chromosomes, has been adapted for the localization of specific DNA sequences in nuclei of Plasmodium berghei. After hybridization using probes labeled with biotin-11-dUTP, the formed DNA/DNA hybrids were detected by fluorescence microscopy using a specific double-layer antibody technique. Besides its high resolution, this procedure is characterized by a high sensitivity, allowing the detection of a unique sequence as small as 2.5 kb.     

Detection of DNA sequences in Plasmodium berghei by means of in situ hybridization

Summary A non-radioactive in situ hybridization technique, used to map unique DNA sequences to plant chromosomes, has been adapted for the localization of specific DNA sequences in nuclei of Plasmodium berghei. After hybridization using probes labeled with biotin-11-dUTP, the formed DNA/DNA hybrids were detected by fluorescence microscopy using a specific double-layer antibody technique. Besides its high resolution, this procedure is characterized by a high sensitivity, allowing the detection of a unique sequence as small as 2.5 kb.     

Identification of trisomy in Macaca fascicularis by fluorescence in situ hybridization with a human chromosome 13 DNA library

A juvenile macaque monkey with abnormal phenotypic and behavioral features was studied cytogenetically. An additional autosome was found in over 90% of the animal's cultured cells. This chromosome, subsequently identified as number 16 in the macaque karyotype by G-banding, was shown to be mostly homologous with human chromosome 13 using fluorescence in situ hybridization of a human chromosome specific cosmid library. Although the monkey, now deceased, exhibited some abnormal physical and behavioral features, none of the severe clinical characteristics associated with human chromosome 13 trisomy were apparent. We suggest that the incomplete expression of 13-trisomy observed could result if the macaque chromosome were deficient in some of the region(s) of chromosome 13 common to humans affected with the disorder.     

Labeling of the centromeric region on human chromosome 8 by in situ hybridization

Summary Probe DNA that binds preferentially to the centromeric region of human chromosomes 8 was synthesized. Alpha satellite probe DNA molecules were selectively amplified from sorter-purified human chromosomes 8 by in vitro DNA amplification using the polymerase chain reaction (PCR). Probe labeling was performed during PCR by incorporation of biotinylated deoxyuridine. In situ hybridization of unpurified probe DNA comprised of alpha satellite monomer and higher molecular weight DNA fragments with metaphase chromosome spreads showed binding to the centromeric regions of numerous chromosomes. However, blocking with unlabeled total human alphoid DNA dramatically reduced crosshybridization to chromosomes other than 8. Under these conditions, the degenerate probe DNA allowed unambiguous visualization of domains occupied by centromeric DNA of chromosome 8 in metaphase spreads and interphase cell nuclei, thus greatly facilitating the detection of numerical chromosome aberrations in tumor cells. In situ hybridization of size-fractionated alpha satellite DNA identified the monomeric fraction as the major cause of crosshybridization. Alpha satellite dimers and higher molecular weight DNA fragments showed relatively high specificity for human chromosomes 8.     

Detection of two mRNA species at single-cell resolution by double-fluorescence in situ hybridization

Here we describe a fluorescence in situ hybridization protocol that allows for the detection of two mRNA species in fresh frozen brain tissue sections. This protocol entails the simultaneous and specific hybridization of hapten-labeled riboprobes to complementary mRNAs of interest, followed by probe detection via immunohistochemical procedures and peroxidase-mediated precipitation of tyramide-linked fluorophores. In this protocol we describe riboprobes labeled with digoxigenin and biotin, though the steps can be adapted to labeling with other haptens. We have used this approach to establish the neurochemical identity of sensory-driven neurons and the co-induction of experience-regulated genes in the songbird brain. However, this procedure can be used to detect virtually any combination of two mRNA populations at single-cell resolution in the brain, and possibly other tissues. Required controls, representative results and troubleshooting of important steps of this procedure are presented. After tissue sections are obtained, the total length of the procedure is 2-3 d.     

Detection of deletions and cryptic translocations in Miller-Dieker syndrome by in situ hybridization.

Fluorescence in situ hybridization (FISH) using two cosmid probes (41A and P13) from the Miller-Dieker syndrome (MDS) critical region in 17p13.3 was performed in a blinded comparison of three MDS patients with submicroscopic deletions and in four normal relatives used as controls. The controls showed both chromosome 17 homologues labeled in 85%-95% of cells, while each patient showed only one homologue labeled in 75%-80% of cells. Two MDS patients with cryptic translocations were also studied. In one case, a patient and her mother had the same der(17) (p+), but the reciprocal product of the translocation could not be identified in the mother by G-banding (i.e., it was a "half-cryptic" translocation). FISH revealed a 3q;17p translocation. The other case involved a patient with apparently normal karyotype. Because a large molecular deletion was found, a translocation involving two G-negative telomeres (i.e., a "full-cryptic" translocation) was postulated. FISH studies on her father and normal brother showed an 8q;17p translocation. These studies demonstrate that in situ hybridization is an efficient method for deletion detection in Miller-Dieker syndrome. More important, parental studies by FISH on patients demonstrating molecular deletions and a normal karyotype may identify cryptic translocation events, which cannot be detected by other molecular genetic strategies. Similar in situ strategies for deletion detection can be developed for other microdeletion syndromes, such as Prader-Willi/Angelman syndrome or DiGeorge syndrome.