Effect of fixation period on HER2/neu gene amplification detected by fluorescence in situ hybridization in invasive breast carcinoma.

This study investigated if formalin fixation duration affects HER2/neu gene amplification detection by fluorescence in situ hybridization (FISH) in breast cancer. Tumor tissues from 35 cases were divided into three groups and subjected to two formalin fixation protocols per group (12 hr, 27 hr in the first; 2 hr, 17.5 hr in the second; 28.5 hr, 541 hr in the third) before FISH analysis. There was no significant difference in FISH signal detection between the two different fixation protocols in the first two groups. In the third, no signal was detected in 4/6 cases fixed for an extended duration.     

Role of bone marrow imprints in haematological diagnosis: a detailed study of 3781 cases

X. Gong, X. Lu, X. Wu, R. Xu, Q. Tang, G. Xu, L. Wang, X. Zhang and X. Zhao Role of bone marrow imprints in haematological diagnosis: a detailed study of 3781 cases Objectives: To explore the role of imprints in routine bone marrow (BM) diagnosis. Methods: The cellularity and diagnostic accuracy of BM imprints, aspirate smears and trephine biopsy sections from 3781 patients were assessed using routine cytochemical staining. Seventy‐nine cases of lymphoma and 114 cases of plasma cell myeloma (PCM) were selected for correlation analysis of tumour cell infiltration patterns. Another 21 cases of lymphoma were selected to detect t(14;18)(q32;q21) and t(11;14)(q13;q32) by fluorescent in situ hybridization (FISH) on BM imprints, and the G‐banding technique was performed for comparison. Results: BM imprints were better than smears for evaluating cellularity. In the BM imprint group, diagnostic accuracy for metastatic carcinoma, myeloproliferative neoplasm, myelodysplastic/myeloproliferative neoplasm and PCM was better than in the smear group, while accuracy for megaloblastic anaemia, acute myeloid leukaemia, refractory cytopenia with unilineage or multilineage dysplasia, refractory anaemia with excess blasts and lymphoplasmacytic lymphoma was higher than in the section group, but not statistically different from the smear group. Good correlation of infiltration patterns of lymphoma and myeloma cells was found between BM imprints and sections (r = 0.90 and 0.78, respectively). Detection of t(11;14)(q13;q32) by FISH on imprints was higher than G‐banding analysis. Conclusions: BM imprints show features of both smears and trephine sections. Imprints are superior to smears for evaluation of cellularity, and are also better than sections for analysis of cytological changes. In addition, FISH on BM imprints markedly improves the identification of chromosomal abnormalities.     

Molecular cytogenetics in metaphase and interphase cells for cancer and genetic research, diagnosis and prognosis. Application in tissue sections and cell suspensions

As the pioneer among molecular cytogenetics techniques, fluorescence in situ hybridization (FISH) allows identification of specific sequences in a structurally preserved cell, in metaphase or interphase. This technique, based on the complementary double-stranded nature of DNA, hybridizes labeled specific DNA (probe). The probe, bound to the target, will be developed into a fluorescent signal. The fact that the signal can be detected clearly, even when fixed in interphase, improves the accuracy of the results, since in some cases it is extremely difficult to obtain mitotic samples. FISH is still used mostly in research, but there are diagnostic applications. New nomenclature is being developed in order to define many of the aberrations that were not distinguished before FISH. Prenatal diagnosis of aneuploidies and malignancies are promptly detected with FISH, which is very useful in critical cases. In some tumors, where chromosomal abnormalities are too complicated to classify manually, the technique of comparative genomic hybridization (CGH), a competitive FISH, allows examiners to determine complete or partial gain or loss of chromosomes. CGH results allow the classification of many tumor cell lines and along with other complementary techniques, like microdissection-FISH, PRINS, etc., increase the possibility of choosing an appropriate treatment for cancer patients.     

Grid technology in tissue-based diagnosis: fundamentals and potential developments

DNA fluorescence in situ hybridization (FISH) technology is used to study chromosomal and genomic changes in fixed cell suspensions and tissue block preparations. The technique is based on specific hybridization of small labeled DNA fragments, the probes, to complementary sequences in a target DNA molecule. Demand for FISH assays in formalin-fixed, paraffin-embedded tissues has been increasing, mainly in conditions in which diagnosis is not achieved in cell smears or tissue imprints, such as solid tumors. Moreover, the development of molecular targeted therapies in oncology has expanded the applicability of tests to predict sensitivity or resistance to these agents. The efficient use of tyrosine kinase inhibitors (TKI) of the epidermal growth factor receptor (EGFR) as therapeutical agents in advanced non-small cell lung cancer (NSCLC) depends on identification of patients likely to show clinical benefit from these specific treatments. The EGFR gene copy number determined by FISH has been demonstrated as an effective predictor of outcome from NSCLC patients to EGFR TKIs; however there are pending challenges for standardization of laboratory procedures and definition of the scoring system. This methodology article focuses on the EGFR FISH assay. It details the scoring system used in the studies conducted at the University of Colorado Cancer Center in which a significant association was found between increased EGFR copy numbers and clinical outcome to TKIs, and proposes interpretative guidelines for molecular stratification of NSCLC patients for TKI therapy.     

Application of high throughput cell array technology to FISH: investigation of the role of deletion of p16 gene in leukemias

Bio-cell chip is a chip that has hundreds of types of cells arrayed and immobilized on a small slide. To elucidate the role of deletion of the p16 gene in hematologic malignancies, the bio-cell chip technique was applied to fluorescent in situ hybridization (FISH) study. We made a bio-cell chip with bone marrow specimen from 109 patients with acute lymphoblastic leukemia (ALL), 102 patients with acute myelogenous leukemia (AML), 47 patients with chronic myelogenous leukemia (CML), and 25 patients with multiple myeloma (MM). A glass slide with 96 separated areas was fabricated, onto which was added methanol/acetic acid fixed cell suspensions for high-throughput FISH for p16. With the successful application of bio-cell chip technique, we found that the deletion of p16 contributed to the oncogenesis in acute leukemia, but not in chronic leukemia. In conclusion, the bio-cell chip, a cell version of ultrahigh-throughput technology, was successfully applied to the FISH study, which can be utilized efficiently in the molecular cytogenetic investigation of hematologic malignancies.     

The specificity of interphase FISH translocation probes in formalin fixed paraffin embedded tissue sections is readily assessed using automated staining and scoring of tissue microarrays constructed from murine xenografts

Implementation of interphase fluorescence in situ hybridization (FISH) assays in the clinical laboratory requires validation against established methods. Validation tools in common use include exchange of consecutive sections with another institution that has already established the FISH assay, comparison with conventional banded metaphase cytogenetics, confirmation of specificity using probed normal metaphases, consecutive paraffin sections of a validation set tested by a reference laboratory, and specificity assessment against well characterized cell lines. We have investigated the feasibility of using tissue microarrays (TMA) constructed from murine xenografts as a preliminary specificity-screening tool for validation of interphase FISH assays. Cell lines currently in use for FISH controls are used to generate xenografts in SCID mice which are fixed in formalin and paraffin embedded. A TMA is constructed using duplicate donor cores from the xenograft blocks. Xenografts used represent a wide range of translocations used routinely for formalin fixed paraffin embedded sections evaluated by FISH. Probe cocktails (Abbott-Vysis), for several non-random translocations associated with hematologic neoplasms and soft tissue sarcomas have been used in this manner. On-line deparaffinization, cell conditioning, and prehybridization steps are automated using a staining workstation (Ventana Discovery XT); hybridization and stringency washes are performed manually offline. FISH-probed TMAs are tracked using a Metasystems image scanner and analyzed using classifiers specifically developed for each molecular abnormality. FISH results for each xenograft in the TMA correspond exactly to the genotype previously established for the parent cell line from which the xenograft was prepared. Moderate complexity tissue microarrays constructed from murine xenografts are excellent validation tools for initial assessment of interphase FISH probe specificity.     

Improvement of combined FISH and immunofluorescence to trace the fate of somatic stem cells after transplantation.

Fluorescence in situ hybridization (FISH) combined with immunohistochemistry of tissue-specific markers provides a reliable method for characterizing the fate of somatic stem cells in transplantation experiments. Furthermore, the association between FISH and fluorescent gene reporter detection can unravel cell fusion phenomena, which could account for apparent transdifferentiation events. However, despite the widespread use of these techniques, they still require labor-extensive protocol adjustments to achieve correct and satisfactory simultaneous signal detection. In the present paper, we describe an improvement of simultaneous FISH and immunofluorescence detection. We applied this protocol to the identification of transplanted human and mouse hematopoietic stem cells in murine brain and muscle. This technique provides unique opportunities for following the path taken by transplanted cells and their differentiation into mature cell types.     

High-level detection of gene amplification and chromosome aneuploidy in extracted nuclei from paraffin-embedded tissue of human cancer using FISH: a new approach for retrospective studies

A novel application of fluorescence in situ hybridization (FISH) to isolated nuclei is described. The method detects gene amplification and chromosome aneuploidy in extracted nuclei from paraffin-embedded tissue of human cancer with greater sensitivity and specificity than existing FISH methods. In this study, the method is applied to signal detection of the HER-2/neu (c-erbB-2) gene, whose amplification is one of the most common genetic alterations associated with human breast cancer. Nuclei were extracted and isolated from formalin fixed, paraffin embedded tissue of 43 different carcinomas (breast, ovary, endometrium, gastrointestinal stromal tumor and malignant mesothelioma). FISH was performed both on sections and extracted nuclei of each tissue using chromosome enumeration probes (CEP) for the centromeric regions of chromosomes 8 and 17, and a locus specific identifier (LSI) for the HER-2/neu oncogene. Differences between ploidy calculated in sections and extracted nuclei were seen in 3 breast carcinomas and 1 gastrointestinal stromal tumor (GIST). Furthermore, 1 breast cancer, previously considered to be borderline for HER-2/neu gene amplification turned out to be clearly amplified. Nuclei extraction and isolation bypass all the problems related to signal interpretation in tissue sections, and the adoption of this new technique, which improves the signal quality in several neoplastic samples, is suggested.     

Immunohistochemistry on Resin Sections: A Comparison of Resin Embedding Techniques for Small Mucosal Biopsies

We have modified resin embedding methods to provide optimal information from en-doscopic biopsies. Mucosal biopsies were fixed either in buffered formalin and processed for embedding in Araldite or in acetone containing protease inhibitors and embedded in glycol meth-acrylate (GMA). GMA embedding generated an im-munophenotypic profile similar to that obtained in frozen sections while yielding far superior morphology and greater numbers of sections from small biopsies. The phenotypic markers included those for T cells, macrophages, mast cells, eosin-ophils and neutrophils. We have also demonstrated collagens, cell adhesion molecules and integrin molecules. Sections of similar quality were obtained with Araldite but the repertoire of antibodies was restricted to those which can be applied to formalin fixed, paraffin embedded tissues. We suggest that for optimal results, small biopsies to be subjected to immunochemistry are fixed in acetone at -20 C with the inclusion of protease inhibitors and embedded in GUIA with careful temperature control.     

The effect of nucleobase-specific fluorescence quenching on in situ hybridization with rRNA-targeted oligonucleotide probes

Oligonucleotide probes labeled with fluorescent dyes are used in a variety of in situ applications to detect specific DNA or RNA molecules. It has been described that probe fluorescence might be quenched upon hybridization in a sequence specific way. Here, a set of 17 oligonuleotides labeled with 6-carboxyfluorescein was used to examine the relevance of nucleotide specific quenching for fluorescence in situ hybridization (FISH) to whole fixed bacterial cells. Probes quenched upon hybridization to a guanine-rich region of purified RNA in solution were not quenched upon FISH. Among other factors the high protein concentration within cells may prevent quenching of probe fluorescence in situ.     

Fluorescent in situ hybridization on tissue microarrays: challenges and solutions

Tissue microarray (TMA) technology has provided a high throughput means of evaluating potential biomarkers and therapeutic targets in archival pathological specimens. TMAs facilitate the rapid assessment of molecular alterations in hundreds of different tumours on a single slide. Sections from TMAs can be used for any in situ tissue analysis, including fluorescent in situ hybridization (FISH). FISH is a molecular technique that detects numerical and structural abnormalities in both metaphase chromosomes and interphase nuclei. FISH is commonly used as a prognostic and diagnostic tool for the detection of translocations and for the assessment of gene deletion and amplification in tumours. Performing FISH on TMAs enables researchers to determine the clinical significance of specific genetic alterations in hundreds of highly characterized tumours. The use of FISH on archival paraffin embedded tissues is technically demanding and becomes even more challenging when applied to paraffin embedded TMAs. The problems encountered with FISH on TMAs, including probe preparation, hybridization, and potential applications of FISH, will be addressed in this review.     

rRNA sequence-based scanning electron microscopic detection of bacteria

A new scanning electron microscopic method was developed for gaining both phylogenetic and morphological information about target microbes using in situ hybridization with rRNA-targeted oligonucleotide probes (SEM-ISH). Target cells were hybridized with oligonucleotide probes after gold labeling. Gold enhancement was used for amplification of probe signals from hybridized cells. The hybridized cells released a strong backscatter electron signal due to accumulation of gold atoms inside cells. SEM-ISH was applied to analyze bacterial community composition in freshwater samples, and bacterial cell counts determined by SEM-ISH with rRNA-targeted probes for major phyla within the domain Bacteria were highly correlated to those by fluorescent in situ hybridization (FISH). The bacterial composition on surface of river sediment particles before and after cell dispersion treatment by sonication was successfully revealed by SEM-ISH. Direct enumeration of bacterial cells on the surface of sonicated sediment particles by SEM-ISH demonstrated that members of Cytophaga-Flavobacterium existed tightly on the surface of particles. SEM-ISH allows defining the number and distribution of phylogenetically defined cells adherent to material surfaces, which is difficult in FISH, and it gives new insight into electron microscopic studies of microorganisms in their natural environment.     

A simple method for the production of bacterial controls for immunohistochemistry and fluorescent in situ hybridization

Immunohistochemical and fluorescent in situ hybridization (FISH) assays are useful diagnostic methods for the identification of bacteria on formalin fixed paraffin embedded histological sections. To validate an anti-bacterial antibody or an oligonucleotide probe and to ensure fidelity during subsequent analyses, suitable positive and negative controls are necessary. Suspensions of fixed bacteria are often used, but ideally, these controls should be fixed, embedded and processed in the same way of tissue samples under analysis. Herein, we describe a simple method for the production of bacterial histological control samples: the sandwich. The sandwich is composed of two external layers of equine lung parenchyma and a central layer of the target bacterium. We prepared sandwiches containing Escherichia coli, Campylobacter jejuni, and Arcanobacterium pyogenes and tested them with appropriate antibodies and Eub338 FISH probe. The sandwich is an effective and simple method to prepare bacterial histological controls fixed and processed in the same way as the diagnostic tissues.     

Improvements in cytogenetic slide preparation: controlled chromosome spreading, chemical aging and gradual denaturing

BACKGROUND: Metaphase spreading is an essential technique for clinical and molecular cytogenetics. Results of classical banding techniques as well as complex fluorescent in situ hybridization (FISH) applications, such as comparative genomic hybridization (CGH) or multiplex FISH (M-FISH), are greatly influenced by the quality of chromosome spreading and pretreatment of the slide prior to hybridization. Materials and Methods Using hot steam and a metal plate with a temperature gradient across its surface, a reproducible protocol for slide preparation, aging, and hybridization was developed. RESULTS: This protocol yields good chromosome spreads from even the most difficult cell suspensions and is unaffected by the environmental conditions. Chromosome spreads were suitable for both banding and FISH techniques common to the cytogenetic laboratory. Chemical aging is a rapid slide pretreatment procedure for FISH applications, which allows freshly prepared cytogenetic slides to be used for in situ hybridization within 30 min, thus increasing analytical throughput and reducing benchwork. Furthermore, the gradually denaturing process described allows the use of fresh biologic material with optimal FISH results while protecting chromosomal integrity during denaturing. CONCLUSION: The slide preparation and slide pretreatment protocols can be performed in any laboratory, do not require specialized equipment, and provide robust results.     

Identification of Nitrite-Reducing Bacteria Using Sequential mRNA Fluorescence In Situ Hybridization and Fluorescence-Assisted Cell Sorting

Sequential mRNA fluorescence in situ hybridization (mRNA FISH) and fluorescence-assisted cell sorting (SmRFF) was used for the identification of nitrite-reducing bacteria in mixed microbial communities. An oligonucleotide probe labeled with horseradish peroxidase (HRP) was used to target mRNA of nirS, the gene that encodes nitrite reductase, the enzyme responsible for the dissimilatory reduction of nitrite to nitric oxide. Clones for nirS expression were constructed and used to provide proof of concept for the SmRFF method. In addition, cells from pure cultures of Pseudomonas stutzeri and denitrifying activated sludge were hybridized with the HRP probe, and tyramide signal amplification was performed, conferring a strongly fluorescent signal to cells containing nirS mRNA. Flow cytometry-assisted cell sorting was used to detect and physically separate two subgroups from a mixed microbial community: non-fluorescent cells and an enrichment of fluorescent, nitrite-reducing cells. Denaturing gradient gel electrophoresis (DGGE) and subsequent sequencing of 16S ribosomal RNA (rRNA) genes were used to compare the fragments amplified from the two sorted subgroups. Sequences from bands isolated from DGGE profiles suggested that the dominant, active nitrite reducers were closely related to Acidovorax BSB421. Furthermore, following mRNA FISH detection of nitrite-reducing bacteria, 16S rRNA FISH was used to detect ammonia-oxidizing and nitrite-oxidizing bacteria on the same activated sludge sample. We believe that the molecular approach described can be useful as a tool to help address the longstanding challenge of linking function to identity in natural and engineered habitats.     

Bacterial communities associated with benthic organic matter in headwater stream microhabitats

Bacterial communities associated with a variety of benthic detritus types were studied in three streams in the context of the chemical characteristics of the sediment material and the stream water. A cell purification assay was developed for a quantitative microscopic evaluation of bacterial community structure in detritus samples by fluorescence in situ hybridization (FISH). The efficiency of FISH with fluorescently monolabelled probes was compared with FISH with signal amplification by catalysed reporter deposition (CARD-FISH). In detritus types poor in organic carbon and nitrogen, the numbers of prokaryotes were related to the chemical characteristics of the stream water column, whereas no such relationship was found for detritus types rich in organic carbon and nitrogen. These results might help to provide criteria for the selection of detritus types for river ecosystem assessment and monitoring. The percentage of bacteria detected by FISH with monolabelled probes was correlated with the detritus total organic matter (OM). This is likely attributed to a higher ribosome content of microbial cells on substrates rich in OM. Cell detection by CARD-FISH did not show any correlation with OM content, indicating that this technique renders the results more independent from the activity state of cells. Fluorescence in situ hybridization with four group-specific probes suggested a relationship between substrate quality and the composition of the microbial assemblages on the various types of detritus. The improved protocol for cell purification and CARD-FISH may facilitate future investigations on the relationship between the riverine benthic detritus quality and microbial community composition.     

Specific cytogenetic labeling of bovine spermatozoa bearing X or Y chromosomes using fluorescent in situ hybridization (FISH)

X and Y specific probes were identified in order to apply the fluorescent in situ hybridization (FISH) technique to bovine spermatozoa. For Y chromosome detection, the BRY4a repetitive probe, covering three quarters of the chromosome, was used. For X chromosome detection, a goat Bacterial Artificial Chromosome (BAC) specific to the X chromosome of bovine and goats and giving a strong FISH signal was used. Each probe labeled roughly 45% of sperm cells. The hybridization method will be useful for evaluating the ratio of X- and Y- bearing spermatozoa in a sperm sample and consequently can be used to evaluate the efficiency of sperm sorting by different techniques such as flow cytometry.     

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.     

Obstacles to flow cytometric analysis of rumen microbial samples

Several methods were tested that would improve the fluorescence signal from hybridized rumen bacterial cells. Disruption of cell envelopes by lysozyme, EDTA, proteinase K and/or SDS caused only a minor increase in fluorescence signal. Use of helper unlabeled oligonucleotide probes was successful only with the Puni[H672] probe which, however, when used with specific PBBl4-labeled probe, gave fluorescence signal drop. No substantial rise in fluorescence signal was also observed with cells subjected to growth-without-cell-division treatment. Further improvements are needed to make the fluorescent in situ hybridization (FISH)-flow cytometry combination applicable to rumen bacteria.