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FLUORESCENCE INSITU HYBRIDIZATION(FISH)

Introduction

Fluorescence In Situ Hybridization (FISH) is a powerful technique for detecting chromosomal changes in tumor cells and is one of the most frequently used techniques in the study of structural cytology of the cell nucleus. It provides a reliable means for studying the genetic composition of cells in mitosis as well as in interphase.

Introduction
When you analyze the modern technologies we can deduce that Single concept has multiple applications. Multiple concepts are incorporated in one technology. One technology has multidisciplinary applications.

Concepts related to fluorescence and fluorescent labeled probes . Concepts related to FISH imaging .
Concepts related to hybridization in molecular biology . Applications of FISH.

CONCEPTS RELATED TO

FLUORESCENCE

History

1602: The cobbler and alchemist V CASCIAROLO prepared by accident an artifical phosphor known as the Bolognian stone or Bolognian phosphor which glows after exposure to light. A complete description of V CASCIAROLOs stone and the first scientific study of lumines- cent phenomena were written in 1640 by F LICETUS (Litheosphorus Sive De Lapide Bono- niensi).
1646: The Jesuit priest A KIRCHER recorded an observation of the wood extract of Lignum nephriticum: an aqueous infusion of this wood exhibited blue color by reflected light and yellow color by transmitted light. The blue light is actually a type of light emission (fluorescence); therefore, A KIRCHER is often regarded as the discoverer of fluorescence. 1852: G STOKES, professor of mathematics and physics, interpreted the light-emitting phenomenon and formulated the law that the fluorescent light is of longer wavelength than the exciting light (Stokes law or Stokes shift). 1853: G STOKES coined the term fluorescence from the term internal dispersion.

History

1935: The physicist A JABLONSKI descibed physically (energy diagrams) the observations made by G Stokes in 1852. 1941: AH COONS and co-workers described in the years 1941 and 1942 the concept of cellular antigen staining in tissue sections by an immunofluorescence technique (fluoresceine -4- isocyanate) for the microscope. 1948: T FRSTER described the physical effects of intermolecular energy migration and fluorescence which are now designated as fluorescence resonance energy transfer (FRET). Today, FRET detection methods are very efficient for probing cells in fluorescence microscopy which allow resolution at the nanometer scale. 1961: M MINSKY designed a confocal microscope (US Patent No. 3,013,467, filed 1957 and awarded 1961).

Definition
Luminescence is most conveniently defined as the radiation emitted by a molecule, or an atom, after it had absorbed energy to go to an exited state.

Fluorescence

Once a molecule arrives at the lowest vibrational level of an exited singlet state, it can do a number of things, one of which is to return to the ground state by photon emission. This process is called fluorescence. The lifetime of an excited singlet state is approximately 10-9 to 10-7sec . Therefore the decay time of fluorescence is of the same order of magnitude. The change in photon energy causes a shift of the fluorescence spectrum to longer wavelength, relative to the absorption spectrum, this is referred to as the Stokes Shift or strokes law .

Fluorescence
A singlet state is one in which all of the electrons in the molecule have their spins paired. Triplet states are those in which one set of electron spin have become unpaired.

Fluorescence

Fluorescence

A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation.
AMCA (amino-methylcoumarin-acetic acid)

Fluorescence

Fluorescence

Rhodamine-, fluorescein-, and coumarin-based dyes cover the three primary colors of the visible part of the electromagnetic spectrum. By using selective excitation and emission Filters, and dichroic mirrors, these colors can be visualized without crosstalk, making triple color FISH feasible. The key advantage of fluorescence tools is their inherently greater sensitivity and range in comparison to methods based upon changes in optical density or chemiluminescent emission; the latter process emits one photon per molecule in comparison to hundreds to thousands of photons emitted by one fluorochrome.

FISH imaging

FISH imaging

Flow cytometry tells us a little about a great many cells whereas the complementary tool of fluorescence microscopy tells us a great deal about only a comparatively few cells.

Flow cytometry, however, is not very informative concerning the spatial locations or dynamics of constituent molecules and cannot be extended to in vivo experiments.

Fluorescence microscopy

Wide field illumination

Conventional fluorescence microscopy is performed with a compound microscope that includes an objective lens and eyepieces. During wide-field imaging, the entire sample is illuminate with excitation light and can be viewed through the oculars or CCDs.

Epi-illumination

Light source mercury bulb Excitation filter it separates the excitation wavelength rays and projects on to the sample through dichroic mirror. Emission filter isolates only rays of emission spectrum and projects on eye or CCD. Dichroic mirror only allows emitted rays through it and blocks excitation rays

Epi-illumination fluorescence microscopic configuration

Confocal microscopy

Confocal microscopy

Hybridization

Principle
The nucleic acid hybridization is the process wherein two DNA or RNA single chains from different biological sources, make the double catenary configuration, based on nucleotide complementarity and of contingent sequence homology of the two sources, resulting DNA-DNA, RNA-RNA or DNA-RNA hybrids. In most cases, the purpose of the hybridization techniques is identification or localization of certain nucleic acid sequences in the genome of some species.

Hybridization

Target molecule represent the DNA, RNA or protein sequence that should be identified.

probe molecules -who identify the target, by hybridization

Stages of Insitu hybridization

Probes

Probe refers to a stretch of nucleotides (from few base pairs to thousands ) that is used to detect a specific region of DNA or RNA as a function of its complemntarity to the target sequence .

Probes

Labeling the probes

Labeling of probes

Types of probes in FISH

SLIDE PREPERATION

Coating the slide with organosilane solution improves tissue adherence from about 10% to 99%

Other methods Gelatin Polylysine Denhardts solution . Most effective is silane coated .

Tissue fixation

Both formalin fixed and frozen tissue sections can be used for ISH . Disadvantage of Frozen sections is poor morphology and pretreatment by proteinases further deteriorates the morphology . Buffered formalin is an excellent fixative for ISH . Hybridization signal depends upon fixation ,using Unbuffered formalin reduces hybridization signal to half . More dramatic reductions are seen with Zenkers and Bouins solution due to presence of mercury and picric acid respectively.

Pretreatment
Pretreatment of the sample is useful for better exposure o target DNA or mRNA. This is done by Pepsin-2mg/ml for 12-25 min Proteinase K of 0.25-1.0ml/ml.

Denaturation

Denaturation of both target DNA and probe DNA is done in combine or in separately. Olignucleotide probes or mRNA does not require this step as they are already single stranded .

This step is influenced by PH of the medium Temperature used . Add 10micro lit of probe to given tissue Place a coverslip Place the slide over hot plate for 95 to 100 c for 5min.

Hybridization

Here we have Target DNA Probe DNA Formamide which facilitate the denaturation of the probe and target . Its concentration is less if we use oligonucleotide probes as they do not require denaturation . Salt solution it should be of low concentration to facilitate denaturation. Dextran sulphate increases effective concentration of probe thus increases rate of hybrid formation. Place the slides in humid chamber 37c for 2 hrs .

Post hybridization
Post hybridization wash is an important step because it removes unwanted probes attached to cell membrane and back ground . 15millmol NaCl, 0.1%bovine serum albumin at 55c-65c for 10min for genomic probes 150millmol Nacl , 0.1%bovine serum albumin -45 to 55c for 10min for oligoprobes .

Factors effecting hybridization


The factors effecting are Temperature Salt concentration Formamide concentration Dextran sulphate Opposite of hybrid is stringency .

Classification of FISH
Types(depending upon the cell cycle) 1. Metaphase FISH
Multiplex Metaphase FISH

2.

Interphase FISH

Applications of FISH

There are many different applications of FISH The major categories of applications include: mapping of genes and other DNA segments in genome research Identification of species-specific chromosomes in somatic cell hybrids Identification of chromosome aberrations, numerical and structural characterization of unknown marker chromosomes.

Microdeletions and Microduplications

Microaberrations or contiguous gene syndromes are caused by the deletion or duplication of genetic material, usually involving multiple conti guous genes on a chromosome. These contiguous gene syndromes, which often involve deletions or duplications that are 2 Mb or less in size, cannot be identified with routine chromosome studies. Therefore, FISH analysis provides a definitive diagnostic test for these disorders.

Cryptic Subtelomeric Rearrangements

It is generally accepted that even with highresolution chromosome analysis, alterations of chromosomal material of less than 24 Mb cannot be detected. Translocations or insertions involving segments below this threshold may be visualized with M-FISH . Abnormalities in the telomeric regions that are not visualized well with G-banding and were historically studied with R- or T-banding, are difficult. Given that these regions are gene rich, they have particular relevance for clinical studies.

Cryptic Subtelomeric Rearrangements

Aberrations of the subtelomeric regions have been documented in a significant percentage of patients with idiopathic mental retardation with an overall frequency of approximately 5%

Duplications and Marker Chromosomes

Approximately 70% of chromosomal duplications are intrachromosomal, whereas 30% involve a nonhomologous chromosome (27). Thus, for the majority of cases, FISH with a single chromosomal library or locus-specific probe from the chromosome with the abnormality will confirm the origin of the duplicated material. There are two basic ways to approach the identification of extra nonhomologous (interchromosomal) material: initial recognition by G-banding and subsequent confirmation with a chromosomal library or locus-specific probe, or multicolor FISH (M-FISH)

Marker chromosomes

Chromosomes that are unidentifiable by routine banding are termed markers. Marker chromosomes represent a heterogeneous group and are typically extra structurally abnormal chromosomes (ESACs).

Marker chromosome

In general, there are two basic methods for delineating the chromosomal origin of marker chromosomes. These include identification by using MFISH or utilizing individual chromosomal libraries or -satellite DNA probes. M-FISH can identify the chromosomal origin of many markers.

Marker chromosomes

Characteristics, such as shape and size of the marker chromosome, determine what probes are best for FISH studies. If the marker is metacentric, it is likely to be an isochromosome and should be studied with satellite probes from chromosomes 8, 9, 12, and 18, as these are the most likely isochromosomes to be present. These are all associated with an abnormal phenotype. If the marker is satellited (or bisatellited), DNA probes from the centromeres of chromosomes 13/21, 14/22, and 15 should be used. These marker chromosomes are associated with mental retardation , cat eye syndrome and turners syndrome .

Prenatal studies

Fluorescence in situ hybridization has been widely used for the detection and analysis of prenatal chromosomal abnormalities. One major advantage of FISH technology is the ability to study uncultured material to produce a rapid result. In addition, FISH is useful for characterizing or detecting subtle abnormalities not delineated by routine banding (e.g., deletions, markers, or duplications).

Ploidy analysis PLOIDY ANALYSIS The vast majority of abnormalities detected prenatally are aneuploidies, involving chromosomes 13, 18, 21, or the sex chromosomes. FISH provides rapid ploidy assessment of these chromosomes by utilizing probes on uncultured interphase cells. In most cases, five probes are used and applied to two different slides (or two different sections of a single slide). All the chromosomes can be visualized by multicolor FISH or Spectral karyotyping

FISH Applications for Studies of Acquired Chromosomal Aberrations


Acute Myelogenous Leukemia Approximately 4060% of acute myelogenous leukemia (AML) patients exhibit genetic aberrations that are easily detected by FISH, and in 2001, the World Health Organization (WHO) established an AML classification system that was based on recurrent genetic abnormalities . For each category, classical cytogenetics identifies the majority of aberrations; however, FISH can be used to detect cryptic abnormalities and variant rearrangements and to monitor disease states during and following treatment. The t(8;21) juxtaposes the AML1 gene on chromosome 21 and the ETO gene on chromosome 8. A dual-color, dual-fusion (DCDF) probe has been developed to detect the fusion products on the derivative 8 and the derivative 21 chromosomes

ALL

Routine cytogenetic studies for acute lymphocytic leukemia (ALL) often produce suboptimal preparations resulting in decreased abnormality detection rates. Therefore, FISH is a useful and necessary adjunct to routine testing. Many clinical laboratories offer an ALL FISH screening panel assessment that might include probes for t(9;22) (BCR/ABL1), MLL, t(12;21), MYC, and a common ALL-associated deletion in 9p21-22 (52,53).

CLL

CHRONIC LYMPHOCYTIC LEUKEMIA/LYMPHOMA Chronic lymphocytic leukemia/lymphoma (CLL) is a chronic lymphoproliferative disorder, primarily of B-cell origin. As a result of the low mitotic rate of affected cells in CLL, metaphase cytogenetics studies only detect genetic aberrations in approximately 40% of cases. Interphase FISH is a more sensitive assay and FISH has largely replaced conventional cytogenetics for the detection of genetic aberrations in CLL. FISH studies reveal that the most common abnormalities include deletions of 13q14, trisomy 12, deletions of 11q22.3-q23.1, deletions of 17p13, and deletions of 6q21q23

PLASMA CELL MYELOMA

PLASMA CELL MYELOMA (MULTIPLE MYELOMA)/PLASMOCYTOMA Chromosomal abnormalities have been reported in approximately 3050% of disorders of plasma cells, and interphase FISH detects deletions and translocations in at least 90% of cases studied (59). Identification of the cytogenetic aberrations has led to the identification of subgroups of plasma cell myeloma with unique clinical and biologic features (60). Translocations involving the Ig heavychain locus (IGH) at 14q32 are frequent and appear to represent early genetic changes. The most common translocations include t(4;14)(p16.3;q32), t(11;14)(q13;q32) (

LYMPHOMA

Non-Hodgkins Lymphoma The genetic hallmarks of many nonHodgkins lymphoma (NHLs) are translocations involving the immunoglobulin (Ig) and T-cell receptor (TCR) genes resulting in inappropriate expression of genes at the reciprocal breakpoints, and FISH presents an effective test for rearrangement assessment.

SOLID TUMORS

Solid Tumors Conventional cytogenetic studies of solid tumors are limited by the ability to culture appropriate cells and to obtain metaphases for chromosome analysis. Analyses from tumors often reveal complex karyotypes with multiple numerical and structural aberrations that might not be well defined by banding. FISH has proven to be a useful tool for detecting abnormalities that allow for proper diagnosis of tumors and/or providing prognostic information. One major advantage of FISH is the ability to study interphase nuclei of touch preparations and paraffinembedded tissue, allowing for assessment of fresh and archival samples.

BREAST CANCER

HER2 and Breast Cancer Amplification of the HER2 (Her-2/neu) gene and/or overexpression of the protein product, which has been demonstrated in approximately 25% of breast cancers, has been associated with poor prognosis, increased risk for recurrence, and shortened survival in breast cancer patients. HER2 assessment is useful for prognosis, chemotherapy responsiveness, and selection for targeted monoclonal antibody therapy (Herceptin) (69). FISH is the most sensitive and specific Food and Drug Administration (FDA)-approved methodology for HER2 detection.

BLADDER CANCER

Bladder Cancer Screening Bladder cancer is a relatively common cancer that has a greater than 70% chance of tumor recurrence (71). A multi-target FISH assay has been developed for diagnosis and for monitoring recurrence of bladder cancer in conjunction with cystoscopy . A panel of probes, consisting of -satellite probes for chromosomes 3, 7, and 17 and a locus-specific probe for 9p21 are used to detect chromosomal aberrations that are commonly associated with bladder cancer (72).

REFERENCES

ANDERSONS PATHOLOGY Molecular Diagnostics For the Clinical LaboratoriesCOLEMAN

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