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Fluorescence in situ hybridization: Uses and limitations
Abstract
The development of molecular hybridization techniques such as fluorescence in situ hybridization (FISH) has had a major Impact on efforts to detect and characterize the genetic changes that give rise to human tumors. With probes designed to Identify specific chromosomes and chromosomal regions, FISH is used routinely by cytogenetics and pathology laboratories to identify recurring chromosomal abnormalities associated with hematologic malignant diseases. In many cases FISH analysis provides increased sensitivity, in that cytogenetic abnormalities have been found In samples that appeared to be normal by morphologic and conventional cytogenetic examination. The combination of cytogenetic, FISH, and molecular analyses provides a powerful approach for diagnosing and subclassifying malignant diseases into clinically and biologically relevant subgroups, In selecting appropriate therapy, and in monitoring the efficacy of therapeutic regimens.
... Due to its significant advantages, FISH technology has greatly contributed to cytogenetic analysis and pathology laboratory's growing potential [5,6]. FISH analysis has high sensitivity, specificity and reproducibility, so the result interpretation relies less on subjective judgement. ...
... There are also limitations of FISH can't be ignored [5,6]. Most notably, as a targeted test, its application is restricted to the abnormalities that are detectable with currently available probes, which limits the utilization as a screening tool for cytogenetically heterogeneous diseases. ...
Fluorescence in situ hybridization (FISH) technique was developed in early 1970s for DNA sequence identification. FISH has gained wide application in cytogenetic research, thanks for its high sensitivity and specificity. FISH analysis has also become a powerful tool for cancer research and treatment, through detecting genetic aberrations in cancer cells. Compared to other cytogenetic tests, FISH analysis is fast and compatible with various cell types. It is particularly useful under clinical settings, by providing crucial information of chromosomal abnormalities, such as gene microdeletion, amplification, and translocation, to guide cancer therapy selection, and monitor effectiveness of the treatment. For example, FISH is used as a companion diagnostic tool for non-small-cell lung cancer (NSCLC) patients with ALK or ROS1 gene rearrangement. It’s also a confirmative test for breast cancer patients with HER2 abnormality. Through combination with other novel technologies, newer versions of FISH have been developed to improve the throughput and sensitivity, which will further expand the potential of FISH analysis in fighting cancer.
... Given that BCas can recur at a frequency of approximately 50% after transurethral resection, early detection, and followup, using minimally invasive techniques are crucial for successful treatment. 20 Cystoscopy is a vital diagnostic tool, but it involves an invasive examination, and some lesions, such as carcinomas in situ, are challenging to detect. 20 Patients often experience discomfort, during the examination, leading to poor compliance. ...
... 20 Cystoscopy is a vital diagnostic tool, but it involves an invasive examination, and some lesions, such as carcinomas in situ, are challenging to detect. 20 Patients often experience discomfort, during the examination, leading to poor compliance. Additionally, the procedure can cause complications such as infection, bleeding, injury, and urethral stricture. ...
Cystoscopy is the current gold standard for diagnosing bladder cancer (Bca), but its invasive nature often results in patient discomfort. This study aims to debise a new strategy to enhance BCa detection. We collected data from 30 BCa patients between January and June 2022. We obtained spontaneously voided urine specimens from these patients before and after administering extracorporeal bladder vibration. These specimens underwent routine cytologic examination and fluorescence in situ hybridization (FISH). Furthermore, we conducted a follow‐up 3 months postoperation. We recollected urine specimens before and after extracorporeal bladder vibration, repeating the cytologic examination and FISH. Our findings indicated an increase in the number of exfoliated cells in patients' urine postvibration compared to previbration. The liquid‐based cell staining results showed an increased detection rate of abnormal cells in the urinary sediment of patients with both low and high‐grade urothelial carcinoma postvibration. Similarly, the FISH results revealed an increased detection rate of CEP3 and CEP7 positive cells postvibration. Additionally, 3 months postoperation, we found abnormal cells in the urine of one patient previbration and in three patients postvibration. Further cystoscopic biopsy confirmed that these three patients had developed tumor recurrence. Our study substantiates that the extracorporeal bladder vibration assay significantly enhances BCa detection and the prediction of tumor recurrence. This method is simple, quick, and cost‐effective, making it a promising approach worthy of widespread clinical application.
... The fluorescently labeled probe locates its matching sequence within the collection of chromosomes and binds to it [66]. The chromosome and sub-chromosomal sites where the fluorescent probe bound can be observed with the aid of a specialized microscope [68]. ...
Wild and domestic cats can benefit effectively from the use of cryopreserved semen. Different breeding techniques have been applied using cryopreserved semen to produce offspring in wild and domestic cats. To date, the success of these techniques in cats is still inadequate. The majority of sperm tend to lose motility and viability following thawing. Semen must be handled carefully, cryopreserved, and thawed by improving current protocols in order to produce post-thaw sperm of acceptable quality and to result in conception after insemination. The quality of sperm obtained following semen cryopreservation process might be beneficial to the success of reproduction performance. To enhance post-thaw recovery of sperm and fertility, it is crucial to have a thorough understanding of the sperm physiology of the species. These factors include the right diluent, sperm dilution, cooling, and thawing rate. Cryopreservation of semen across cat species often results in poor post-thawed sperm quality, including a decrease in motility and acrosomal integrity of up to 30–50% and a rapid decrease in progressive motility within hours of thawing. Due to the low number of sperm and compromised post-thawed sperm quality, laparoscopic-assisted reproduction is frequently used for artificial insemination procedures in Felids.
... Probes can be tailored to the detection of a particular DNA/RNA segment, enhancing diagnostic sensitivity and specificity [11]. However, creating new probes for otherwise unexplored targets requires careful design and evaluation, with the need for rigorous checks and confirmation prior to use [26]. Even when Eubacterial probes are being used to screen for intracellular bacteria, it is important to be cognizant that certain bacterial walls may not allow penetration without the inclusion of enzymatic pre-digestion [27][28][29]. ...
Simple Summary
Bacterial infections have traditionally been identified by culture; however, with advances in the understanding of the role of bacteria in disease, it is recognized that intracellular bacteria can be visualized within cells. Fluorescent in situ hybridization (FISH) is a technique used to not only identify intracellular organisms but also allows researchers to see them, in situ. In this review, we discuss the use of FISH for the identification of intracellular bacteria in companion animals. Most studies have focused on the identification of Escherichia coli, particularly within the gastrointestinal tract. Additionally, bacterial associations with inflammatory diseases affecting the liver, kidney, skin, and cardiovascular systems have been investigated with mixed results.
Abstract
FISH techniques have been applied for the visualization and identification of intracellular bacteria in companion animal species. Most frequently, these techniques have focused on the identification of adhesive-invasive Escherichia coli in gastrointestinal disease, although various other organisms have been identified in inflammatory or neoplastic gastrointestinal disease. Previous studies have investigated a potential role of Helicobacter spp. in inflammatory gastrointestinal and hepatic conditions. Other studies evaluating the role of infectious organisms in hepatopathies have received some attention with mixed results. FISH techniques using both eubacterial and species-specific probes have been applied in inflammatory cardiovascular, urinary, and cutaneous diseases to screen for intracellular bacteria. This review summarizes the results of these studies.
... These properties allow in situ hybridization to be used not only for transcript localization analysis, but also for verification of specificity of newly developed antibodies in combination with immunostaining [19,30]. However, compared to immunostaining, in situ hybridization is a time-consuming procedure with many steps, which is one of the reasons why its clinical application is limited to a few purposes, such as diagnosing chromosomal aberrations [6,15]. ...
In situ hybridization (ISH), which visualizes nucleic acids in tissues and cells, is a powerful tool in histology and pathology. Over 50 years since its invention, multiple attempts have been made to increase the sensitivity and simplicity of these methods. Therefore, several highly sensitive in situ hybridization methods have been developed that offer researchers a wide range of options. When selecting these in situ hybridization variants, their signal-amplification principles and characteristics must be understood. In addition, from a practical point of view, a method with good monetary and time-cost performance must be chosen. This review introduces recent high-sensitivity in situ hybridization variants and presents their principles, characteristics, and costs.
... FISH is a classical and indispensable method for molecular biology research and precise diagnosis 37 . Despite the recent upgrade of this technology, the new generation of cutting-edge FISH technology is still in its infancy. ...
In the unprecedented single-cell sequencing and spatial multiomics era of biology, fluorescence in situ hybridization (FISH) technologies with higher sensitivity and robustness, especially for detecting short RNAs and other biomolecules, are greatly desired. Here, we develop the robust multiplex π-FISH rainbow method to detect diverse biomolecules (DNA, RNA, proteins, and neurotransmitters) individually or simultaneously with high efficiency. This versatile method is successfully applied to detect gene expression in different species, from microorganisms to plants and animals. Furthermore, we delineate the landscape of diverse neuron subclusters by decoding the spatial distribution of 21 marker genes via only two rounds of hybridization. Significantly, we combine π-FISH rainbow with hybridization chain reaction to develop π-FISH+ technology for short nucleic acid fragments, such as microRNA and prostate cancer anti-androgen therapy-resistant marker ARV7 splicing variant in circulating tumour cells from patients. Our study provides a robust biomolecule in situ detection technology for spatial multiomics investigation and clinical diagnosis. Fluorescence in situ hybridization (FISH) methods with high sensitivity are needed. Here the authors develop multiplex πFISH rainbow to detect a range of biomolecules; they also combine this with the hybridization chain reaction to develop πFISH+ technology for short nucleic acid fragment detection.
Cell therapies as potential treatments for Parkinson’s disease first gained traction in the 1980s, owing to the clinical success of trials that used transplants of foetal midbrain dopaminergic tissue. However, the poor standardization of the tissue for grafting, and constraints on its availability and ethical use, have hindered this treatment strategy. Recent advances in stem-cell technologies and in the understanding of the development of dopaminergic neurons have enabled preclinical advancements of promising stem-cell therapies. To move these therapies to the clinic, appropriate levels of safety screening, as well as optimization of the cell products and the scalability of their manufacturing, will be required. In this Review, we discuss how challenges pertaining to cell sources, functional and safety testing, manufacturing and storage, and clinical-trial design are being addressed to advance the translational and clinical development of cell therapies for Parkinson’s disease. This Review discusses requirements—in cell sources, functional and safety testing, manufacturing and storage, and clinical-trial design—for the clinical advancement of cell therapies for Parkinson’s disease.
Fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) is a key biochemical method for the comparative analysis of complex protein mixtures. The technique focuses on the identification and characterization of individual protein species following gel electrophoretic separation making it an important analytical tool of top-down proteomics. In order to verify changes in the expression levels of a particular protein, as determined by 2D-DIGE analysis, and evaluate the subcellular localization of the proteoform of interest, immunofluorescence microscopy is very well suited. This chapter describes in detail the preparation of tissue specimens and the process of cryo-sectioning, as well as incubation with primary antibodies and fluorescently labeled secondary antibodies, followed by image analysis. As illustrative examples, the co-detection of immuno-labeled dystrophin and the Y-chromosome in skeletal muscle are shown, and the localization of calbindin in the cerebellum is presented.
In multiple myeloma, karyotypic 14q32 translocations have been identified at a variable frequency (10–60% in different studies). In the majority of cases, the partner chromosome has not been identified (14q+), and in the remaining cases, a diverse array of chromosomal partners has been implicated, with 11q13 being the most common. We developed a comprehensive Southern blot assay to identify and distinguish different kinds of immunoglobulin heavy chain (IgH) switch recombination events. Illegitimate switch recombination fragments (defined as containing sequences from only one switch region) are potential markers of translocation events into IgH switch regions and were identified in 15 of 21 myeloma cell lines, including seven of eight karyotyped lines that have no detectable 14q32 translocation. From all nine lines or tumor samples analyzed further, cloned illegitimate switch recombination fragments were confirmed to be IgH switch translocation breakpoints. In three of these cases, the translocation breakpoint was shown to be present in the primary tumor. These translocation breakpoints involve six chromosomal loci: 4p16.3 (two lines and the one tumor); 6; 8q24.13; 11q13.3 (in three lines); 16q23.1; and 21q22.1. We suggest that translocations into the IgH locus (i) are frequent (karyotypic 14q32 translocations and/or illegitimate switch recombination fragments are present in primary tumor samples and in 19 of 21 lines that we have analyzed); (ii) occur mainly in switch regions; and (iii) involve a diverse but nonrandom array (i.e., frequently 11q13 or 4p16) of chromosomal partners. This appears to be the most frequent genetic abnormality in multiple myeloma.
Numerical chromosome aberrations were detected in hematological cancers by nonradioactive in situ hybridization (ISH) procedures, using centromere specific probes for chromosomes 1, 7, 8, 9, 10, 11, 16, 17, 18, X, and Y. All 15 cases could be evaluated by ISH for these 11 probes. Our experiments show that in seven of these randomly selected leukemia bone marrow cell suspensions numerical aberrations for one or two chromosomes could be detected by this method. The results of ISH on interphase nuclei and in some cases on metaphase preparations were compared with karyotyping data. Seven cases of chromosomal aberrations observed with ISH (three for monosomy and four for trisomy) were confirmed by this classical cytogenetic technique, whereas in five instances an aberration was found only with ISH (twice for monosomy, twice for trisomy, and one disomy for the Y-probe). One case of a trisomy for chromosome 1 observed by ISH on interphase nuclei could be explained by a marker chromosome, a finding that was further substantiated by ISH on metaphase spreads. In this case double-target ISH on interphase cells with the probes for chromosomes 1 and 16 strongly suggested a translocation between these chromosomes. Also, in one case a marker chromosome could be characterized as a translocation between chromosomes 7 and 17. In this latter case the cytogenetic examinations revealed only monosomy for chromosomes 7 and 17 in addition to noncharacterized marker chromosomes. Our results indicate that the nonradioactive ISH procedure in combination with chromosome specific repetitive centromeric probes is a powerful tool for studying both numerical and structural chromosomal aberrations in interphase nuclei of leukemias. It may therefore become a valuable and routine diagnostic tool in addition to the existing karyotyping procedures.
Chromosomes can be specifically stained in metaphase spreads and interphase nuclei by in situ hybridization with entire chromosome-specific DNA libraries. Unlabeled human genomic DNA is used to inhibit the hybridization of sequences in the library that bind to multiple chromosomes. The target chromosome can be made at least 20 times brighter per unit length than the others. Trisomy 21 and translocations involving chromosome 4 can be detected in metaphase spreads and interphase nuclei by using this technique.
Human chromosomes terminate with specialized telomeric structures including the simple tandem repeat (TTAGGG)n and additional complex subtelomeric repeats1−3. Unique sequence DNA for each telomere is located 100−300 kilobases (kb) from the end of most chromosomes. A high concentration of genes4 and a number of candidate genes for recognizable syndromes5−8 are known to be present in telomeric regions. The human telomeric regions represent a major diagnostic challenge in clinical cytogenetics, because most of the terminal bands are G negative, and cryptic deletions and translocations in the telomeric regions are therefore difficult to detect by conventional cytogenetic methods. In fact, several submicroscopic chromosomal abnormalities in patients with undiagnosed mental retardation or multiple congenital anomalies have been identified by other molecular methods such as DNA polymorphism analysis9,10. To improve the sensitivity for deletion detection and to determine whether such cryptic rearrangements represent a significant source of human pathology that has not been previously appreciated, it would be valuable to have specific FISH probes for all human telomeres. We report here the isolation and characterization of a complete set of specific FISH probes representing each human telomere. As most of these clones are at a known distance of within 100−300 kb from the end of the chromosome arm, this provides a 10-fold improvement in deletion detection sensitivity compared with high-resolution cytogenetics (2−3 Mb resolution). While testing these probes, we serendipitously identified a family with multiple members carrying a cryptic 1q;11p rearrangement in the balanced or unbalanced state.
The t(14;19)(q32.3;q13.1) is a recurring translocation found in the neoplastic cells of some patients with chronic lymphocytic leukemia (CLL) or other B-lymphocytic neoplasms. We previously cloned the translocation breakpoint junctions present in the leukemic cells from three such patients and identified a gene, BCL3, whose transcription is increased as a result of the translocation. In the present paper, we describe three additional patients with the t(14;19), one with lymphoma and two with CLL, and report the cloning and sequencing of the breakpoint junction in one of these patients as well as in a previously reported patient. We and others have found that the breakpoints on chromosome 14, with one exception, fall within the switch region upstream of the immunoglobulin heavy chain Cα1 or Cα2 sequences. Several of the breaks within chromosome 19 fall immediately upstream of the BCL3 gene, but several others are more than 16 kb 5′ of the gene. Most patients with CLL and the t(14;19) also show trisomy 12. Genes Chromosom. Cancer 20:64–72, 1997. © 1997 Wiley-Liss, Inc.
Cytogenetic analysis of unstimulated short-term bone marrow cell cultures was performed on 280 patients with multiple myeloma and related disorders. In 65% of the cases, an additional short term B-cell stimulated culture was also examined. Chromosomally abnormal clones were found in 31% of the patients, 15% in Waldenström macroglobulinemia, 25% in monoclonal gammopathies, 33% in multiple myeloma, and 50% in plasma cell leukemia. Three primary chromosomal breakpoints were recurrently involved: 14q32, 16q22, and 22q11. Structural rearrangements of chromosome I were the most frequent (26% of the abnormal cases), but always as a secondary change. Rearrangements of band 14q32 were found in 22% of the abnormal cases. Among the multiple myeloma patients who showed an abnormal karyotype, 33 (46%) were hyperdiploid, most frequently with 52–56 chromosomes, 29 patients (40%) were pseudodiploid, and the remaining 12 cases (14%) were hypodiploid. A highly significant relation was observed between the presence of an abnormal karyotype and the following clinical parameters: stage III (P = 0.0001), bone marrow plasma cell infiltration greater than 30% (P = 0.0001), presence of bone lesions (P = 0.0009), and β2-microglobulin levels greater than 4 mg/L (P = 0.0001). Genes Chromosom. Cancer 18:84–93, 1997. © 1997 Wiley-Liss, Inc.
We have studied the retinoblastoma (RB-1) susceptibility gene status and pRB expression in 22 human myeloma cell lines (HMCL) and in 10 patients with advanced multiple myeloma (MM). Deletions of the RB-1 gene were observed in 81% (17/21) of the informative HMCL, regardless of their paracrine or autocrine interleukin-6 (IL-6) status. Among the deleted HMCL, only one (U266) had a biallelic deletion and lacked pRB expression. Monoallelic deletions had no consequence on the RB-1 gene activation and pRB expression. One patient of 10 presented the same biallelic deletion as U266 and six of 10 had monoallelic deletions. We conclude that monoallelic deletions of the RB-1 gene are frequent in HMCL and MM patients but have no consequence on gene activation and pRB expression.
Chronic myelogeneous leukemia (CML) is genetically characterized by fusion of the bcr and abl genes on chromosomes 22 and
9, respectively. In most cases, the fusion involves a reciprocal translocation t(9;22)(q34;q11), which produces the cytogenetically
distinctive Philadelphia chromosome (Ph1). Fusion can be detected by Southern (DNA) analysis or by in vitro amplification
of the messenger RNA from the fusion gene with polymerase chain reaction (PCR). These techniques are sensitive but cannot
be applied to single cells. Two-color fluorescence in situ hybridization (FISH) was used with probes from portions of the
bcr and abl genes to detect the bcr-abl fusion in individual blood and bone marrow cells from six patients. The fusion event
was detected in all samples analyzed, of which three were cytogenetically Ph1-negative. One of the Ph1-negative samples was
also PCR-negative. This approach is fast and sensitive, and provides potential for determining the frequency of the abnormality
in different cell lineages.
A method for multiple fluorescence in situ hybridization is described allowing the simultaneous detection of more than three target sequences with only three fluorescent dyes (FITC, TRITC, AMCA), respectively emitting in the green, red, and blue.
This procedure is based on the labeling of (DNA) probes with more than one hapten and visualisation in multiple colors. The possibility to detect multiple targets simultaneously is important for prenatal diagnosis and the detection of numerical and/or structural chromosome aberrations in tumor diagnosis. It may form the basis for an in situ hybridization based chromosome banding technique.