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This article is from The Indian Journal of Medical Research , volume 135 . Abstract Background & objectives:: Fluorescence in situ hybridization (FISH) is increasingly being recognized as the most accurate and predictive test for HER2/neu gene amplification and response to therapy in breast cancer. In the present study we investigated HER-2/neu gene amplification by FISH in breast carcinoma tissue specimens and compared the results with that of immunohistochemical (IHC) analysis. Methods:: A total of 90 breast carcinoma tissue samples were used for immunohistochemical (IHC) and FISH analysis. IHC was performed by using mouse monoclonal antibody to the intracellular domain of HER-2/neu protein. Each slide was scored in a blinded fashion by two pathologists according to the manufacturer's recommended criteria. FISH analysis was performed on paraffin embedded breast tumour tissue sections. The polysomy for centromere 17 (Spec green signal) was read as green signals less than 4 as moderate polysomy, and more than 4 as highly polysomy. Results:: Thirty of the 90 patients had negative results by IHC and FISH. Of the 28 patients with the score of 2+ by IHC, 20 were FISH positive for HER-2/neu gene amplification, three were FISH negative and five patients showed equivocal (1.8-2.2) results by FISH. These five cases were retested for IHC and FISH on different paraffin embedded tissue blocks, and all five were found positive for HER-2/neu gene amplification. Twenty five patients with the score of 3+ by IHC were FISH positive for HER-2/neu gene amplification (>2.2). Seven cases with the score of 3+ by IHC were FISH negative for HER-2/neu gene amplification (>2.2), and showed polysomy of chromosome number 17 high polysomy > 4. Interpretation & conclusions:: Our results indicated that HER-2/neu status by FISH should be performed in all cases of breast tumour with a 2+ score by IHC. Cases demonstrating a 3+ score by IHC may be subjected to FISH to rule out polysomy of chromosome 17 which could be falsely interpreted as HER-2/neu overexpression by IHC analysis. There is also a need for establishing a clinically validated cut-off value for HER-2/neu FISH amplification against IHC which may be further compared and calibrated.

This article is from The Indian Journal of Medical Research , volume 135 . Abstract Background & objectives:: Fluorescence in situ hybridization (FISH) is increasingly being recognized as the most accurate and predictive test for HER2/neu gene amplification and response to therapy in breast cancer. In the present study we investigated HER-2/neu gene amplification by FISH in breast carcinoma tissue specimens and compared the results with that of immunohistochemical (IHC) analysis. Methods:: A total of 90 breast carcinoma tissue samples were used for immunohistochemical (IHC) and FISH analysis. IHC was performed by using mouse monoclonal antibody to the intracellular domain of HER-2/neu protein. Each slide was scored in a blinded fashion by two pathologists according to the manufacturer's recommended criteria. FISH analysis was performed on paraffin embedded breast tumour tissue sections. The polysomy for centromere 17 (Spec green signal) was read as green signals less than 4 as moderate polysomy, and more than 4 as highly polysomy. Results:: Thirty of the 90 patients had negative results by IHC and FISH. Of the 28 patients with the score of 2+ by IHC, 20 were FISH positive for HER-2/neu gene amplification, three were FISH negative and five patients showed equivocal (1.8-2.2) results by FISH. These five cases were retested for IHC and FISH on different paraffin embedded tissue blocks, and all five were found positive for HER-2/neu gene amplification. Twenty five patients with the score of 3+ by IHC were FISH positive for HER-2/neu gene amplification (>2.2). Seven cases with the score of 3+ by IHC were FISH negative for HER-2/neu gene amplification (>2.2), and showed polysomy of chromosome number 17 high polysomy > 4. Interpretation & conclusions:: Our results indicated that HER-2/neu status by FISH should be performed in all cases of breast tumour with a 2+ score by IHC. Cases demonstrating a 3+ score by IHC may be subjected to FISH to rule out polysomy of chromosome 17 which could be falsely interpreted as HER-2/neu overexpression by IHC analysis. There is also a need for establishing a clinically validated cut-off value for HER-2/neu FISH amplification against IHC which may be further compared and calibrated.

This article is from Journal of Visualized Experiments : JoVE . Abstract Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

This article is from Biomarker Research , volume 2 . Abstract Extensive studies of the genetic aberrations related to human diseases conducted over the last two decades have identified recurrent genomic abnormalities as potential driving factors underlying a variety of cancers. Over the time, a series of cutting-edge high-throughput genetic tests, such as microarrays and next-generation sequencing, have been developed and incorporated into routine clinical practice. Although it is a classical low-throughput cytogenetic test, fluorescence in situ hybridization (FISH) does not show signs of fading; on the contrary, it plays an increasingly important role in detecting specific biomarkers in solid and hematologic neoplasms and has therefore become an indispensable part of the rapidly developing field of personalized medicine. In this article, we have summarized the recent advances in FISH application for both de novo discovery and routine detection of chromosomal rearrangements, amplifications, and deletions that are associated with the pathogenesis of various hematopoietic and non-hematopoietic malignancies. In addition, we have reviewed the recent developments in FISH methodology as well.

This article is from The EPMA Journal , volume 5 . Abstract None

This article is from The EPMA Journal , volume 5 . Abstract None

This article is from The EPMA Journal , volume 5 . Abstract None

This article is from The EPMA Journal , volume 5 . Abstract None

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6. Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.

Abnormalities in chromosomes were assessed using cytogenetic and molecular cytogenetic analyses utilizing the FISH technique on blood samples from patients diagnosed with chronic lymphocytic leukemia. The study included the selection of 50 patients (32 males and 18 females) for both the early diagnosis phase (before therapy) and the treatment phase, examining various factors such as sex, age, and occupation. The results revealed that most patients in the over-70 age demographic are male. The majority lacked a familial history of this condition. Patients with chronic lymphocytic leukemia exhibited a higher prevalence of the chromosomal defect deletion (13) (q14) at 43.8%, followed by deletion (11) (q23) at 18.8%. A trisomy 12 alteration was observed at 12.5%, along with a loss on chromosome 17, also present at 12.5%. Tetraploidy occurred seldom (6.2%), notwithstanding the existence of chromosomal defects, specifically deletions (6) at q25-q27. The current study indicates that structural chromosomal modifications were more prevalent than numerical changes regarding chromosomal aberrations, with both types associated with chronic lymphocytic leukemia.

Abnormalities in chromosomes were assessed using cytogenetic and molecular cytogenetic analyses utilizing the FISH technique on blood samples from patients diagnosed with chronic lymphocytic leukemia. The study included the selection of 50 patients (32 males and 18 females) for both the early diagnosis phase (before therapy) and the treatment phase, examining various factors such as sex, age, and occupation. The results revealed that most patients in the over-70 age demographic are male. The majority lacked a familial history of this condition. Patients with chronic lymphocytic leukemia exhibited a higher prevalence of the chromosomal defect deletion (13) (q14) at 43.8%, followed by deletion (11) (q23) at 18.8%. A trisomy 12 alteration was observed at 12.5%, along with a loss on chromosome 17, also present at 12.5%. Tetraploidy occurred seldom (6.2%), notwithstanding the existence of chromosomal defects, specifically deletions (6) at q25-q27. The current study indicates that structural chromosomal modifications were more prevalent than numerical changes regarding chromosomal aberrations, with both types associated with chronic lymphocytic leukemia.

Abnormalities in chromosomes were assessed using cytogenetic and molecular cytogenetic analyses utilizing the FISH technique on blood samples from patients diagnosed with chronic lymphocytic leukemia. The study included the selection of 50 patients (32 males and 18 females) for both the early diagnosis phase (before therapy) and the treatment phase, examining various factors such as sex, age, and occupation. The results revealed that most patients in the over-70 age demographic are male. The majority lacked a familial history of this condition. Patients with chronic lymphocytic leukemia exhibited a higher prevalence of the chromosomal defect deletion (13) (q14) at 43.8%, followed by deletion (11) (q23) at 18.8%. A trisomy 12 alteration was observed at 12.5%, along with a loss on chromosome 17, also present at 12.5%. Tetraploidy occurred seldom (6.2%), notwithstanding the existence of chromosomal defects, specifically deletions (6) at q25-q27. The current study indicates that structural chromosomal modifications were more prevalent than numerical changes regarding chromosomal aberrations, with both types associated with chronic lymphocytic leukemia.

This article is from Scientific Reports , volume 4 . Abstract Molecular biology techniques such as PCR constitute powerful tools for the determination of the taxonomic origin of bones. DNA degradation and contamination by exogenous DNA, however, jeopardise bone identification. Despite the vast array of techniques used to decontaminate bone fragments, the isolation and determination of bone DNA content are still problematic. Within the framework of the eradication of transmissible spongiform encephalopathies (including BSE, commonly known as “mad cow disease”), a fluorescence in situ hybridization (FISH) protocol was developed. Results from the described study showed that this method can be applied directly to bones without a demineralisation step and that it allows the identification of bovine and ruminant bones even after severe processing. The results also showed that the method is independent of exogenous contamination and that it is therefore entirely appropriate for this application.

This article is from Microbes and Environments , volume 27 . Abstract The diversity of cultivable methane-oxidizing bacteria (MOB) in the rice paddy field ecosystem was investigated by combined culture-dependent and fluorescence in situ hybridization (FISH) techniques. Seven microsites of a Japanese rice paddy field were the focus of the study: floodwater, surface soil, bulk soil, rhizosphere soil, root, basal stem of rice plant, and rice stumps of previous harvest. Based on pmoA gene analysis and transmission electron microscopy (TEM), four type I, and nine type II MOB isolates were obtained from the highest dilution series of enrichment cultures. The type I MOB isolates included a novel species in the genus Methylomonas from floodwater and this is the first type I MOB strain isolated from floodwater of a rice paddy field. In the type I MOB, two isolates from stumps were closely related to Methylomonas spp.; one isolate obtained from rhizosphere soil was most related to Methyloccocus-Methylocaldum-Methylogaea clade. Almost all the type II MOB isolates were related to Methylocystis methanotrophs. FISH confirmed the presence of both types I and II MOB in all the microsites and in the related enrichment cultures. The study reported, for the first time, the diversity of cultivable methanotrophs including a novel species of type I MOB in rice paddy field compartments. Refining growth media and culture conditions, in combination with molecular approaches, will allow us to broaden our knowledge on the MOB community in the rice paddy field ecosystem and consequently to implement strategies for mitigating CH4 emission from this ecosystem.

This article is from Microbes and Environments , volume 27 . Abstract The diversity of cultivable methane-oxidizing bacteria (MOB) in the rice paddy field ecosystem was investigated by combined culture-dependent and fluorescence in situ hybridization (FISH) techniques. Seven microsites of a Japanese rice paddy field were the focus of the study: floodwater, surface soil, bulk soil, rhizosphere soil, root, basal stem of rice plant, and rice stumps of previous harvest. Based on pmoA gene analysis and transmission electron microscopy (TEM), four type I, and nine type II MOB isolates were obtained from the highest dilution series of enrichment cultures. The type I MOB isolates included a novel species in the genus Methylomonas from floodwater and this is the first type I MOB strain isolated from floodwater of a rice paddy field. In the type I MOB, two isolates from stumps were closely related to Methylomonas spp.; one isolate obtained from rhizosphere soil was most related to Methyloccocus-Methylocaldum-Methylogaea clade. Almost all the type II MOB isolates were related to Methylocystis methanotrophs. FISH confirmed the presence of both types I and II MOB in all the microsites and in the related enrichment cultures. The study reported, for the first time, the diversity of cultivable methanotrophs including a novel species of type I MOB in rice paddy field compartments. Refining growth media and culture conditions, in combination with molecular approaches, will allow us to broaden our knowledge on the MOB community in the rice paddy field ecosystem and consequently to implement strategies for mitigating CH4 emission from this ecosystem.