Fluorescent In-Situ Hybridisation: Current Applications and Recent Advances
The applications of fluorescent in-situ hybridisation (FISH) as a cytological tool has pervaded across many scientific fields including the medical setting, in areas of prenatal genetic diagnosis, tumour cytogenetics and pathogens. Its applications also extend from toxicology and forensics to food microbiology and plant sciences. Despite being developed over 40 years ago, FISH remains a popular and invaluable fluorescence technique, (Gall & Pardue, 1969). Overtime this technique has been continually developed and progressed to the point where now a single base may now be visualised (Beliveau et al., 2015).
This paper will first discuss the technical aspects that facilitate FISH, including the mechanism of hybridization and DNA sequence visualisation. The remainder of the paper will be a discussion of select applications of FISH, with focus on its biomedical uses.
Mechanism of FISH
In situ hybridization
In situ hybridization facilitates the visualisation of specific nucleic acid sequences, in its native state, by hybridizing or binding complementary DNA or RNA sequences on a probe to the target sequence. These probe sequences can be labelled with radioisotopes or fluorescent molecules. Radioisotopes were used in the initial development of this technique in the 1960’s (Gall & Pardue, 1969). Nowadays, fluorescent molecules are overwhelmingly preferred, with main advantages being its speed and superior signal resolution, along with its ease of use, stability, safety and multiplicity of labels (Swiger & Tucker, 1996).
The basic protocol of FISH, as seen in Figure 1. involves the preparation of target as well as the preparation probe DNA. Target DNA is prepared by fixing onto a slide, commonly with formalin. Next the double stranded target DNA is denatured in the presence of heat, alkaline, low salt concentration and formamide. Probe DNA is labelled with a fluorochrome and then similarly denatured. Probe and target DNA are then hybridized together, followed by washing.
Hybridization is a dynamic reaction allowing a single stranded DNA sequence to return to its native, double stranded confirmation. There are many factors affecting success of hybridization. One factor is the melting temperature (Tm) of the DNA strand, as hybridization occurs just below Tm. Tm itself is dependent on base composition and length. Other important factors include the concentration of probe and target DNA sequences, pH and salt concentration. Hybridization typically involves a formamide solution, as it lowers the Tm. Hybridization also involves saline sodium citrate as sodium ion electrostatically interacts with the negative phosphate DNA backbone (Schildkraut, 1965).
Washing is necessary to remove unbound ssDNA and DNA and non-specific DNA such as probe:probe, target:target complements. The hybridized probe:target complement is able to withstand washing due to its stability and high degree of complementarity.
Probes can be labelled either directly or indirectly with fluorochromes. Direct labelling involves a covalent bonding of DNA and fluorochromes such as fluorescein isothiocyanate (FITC) or rhodamine (Wiegant et al., 1993). The main advantage of direct labelling is its more time efficient nature and saves in the cost of reagents. However, indirect labelling, a more common method of visualisation, is where a probe is bonded to a non-fluorescent label, or hapten such as biotin (Pinkel, Straume, & Gray, 1986)..These bound probes are detected with macromolecular reporter molecules that are coupled to a fluorescent tag. For example, biotin is commonly visualised with avidin coupled to FITC (Swiger & Tucker, 1996). The main advantage of indirect labelling is that it allows for the signal to be amplified, by addition of layers of fluorescent tags.
Furthermore, the signal may be further enhanced using dyes and antifade agents. A commonly used dye is 4′,6-diamidino-2-phenylindole (DAPI), which has a great affinity to DNA as it binds in the DNA minor groove. Antifade agents are used to prolong intensity of fluorescence emission from fluorochromes, which will rapidly fade upon UV light exposure (Johnson & de C. Nogueira Araujo, 1981).
A more in depth protocol can be found at (“Fluorescence in situ hybridization,” 2005).
Figure 1. Basic FISH protocol (Liehr, 2008)
The viewing a hybridized sample involves the use of a fluorescent microscope. The basic components of a fluorescent microscope include a high-pressure mercury lamp to produce short wavelengths of light and an excitation filter to select for these short wavelengths that are characteristic to the fluorochrome. As light reaches the fluorescent hybridized specimen its subsequently emits long wavelengths of light, allowing a barrier filter to transmit these long wavelengths whilst filtering out any short background wavelengths. Hence a fluorescent specimen will appear visible (Liehr, 2008).
Better fluorescence may be obtained when the colour of the fluorochromes contrasts sufficiently, such as a green and orange fluorochrome. Images are also able to be improved throughout stages of visualisation, such as using charged couple device (CCD) in a digital camera or post-image capturing manipulation. Nowadays, multi-bandpass filters can be used, allowing for simultaneous visualisation of multiple fluorochromes (Tanner et al.). This ultimately allows visualisation of specific DNA and RNA sequences, serving as an invaluable biomedical tool.
Prenatal Genetic Abnormality Diagnosis
The first biomedical application of FISH was in 1988 used to detect chromosomal abnormalities, or aneuploidies, for chromosome 21(Pinkel et al., 1988). Since then, other prenatal chromosomal abnormalities have been elucidated and the diagnosis of prenatal genetic abnormalities is important for the prevention of diagnosis and detection of aneuploidy related birth defects, such as down syndrome, trisomy 13 or trisomy 18.
Overtime, FISH has proven to be a both a reliable and an accurate method for prenatal genetic diagnosis. It was FDA cleared in 1997 to test for chromosomes 13, 18, 21, X and Y and has a 99.9% sensitivity, and 100% specificity. It also has a 99% concordance rate 99% with standard cytogenetic karyotyping (Tepperberg et al., 2001).
FISH is advantageous over classical cytogenetic analysis as it much more efficient. Classical cytogenetic analysis involves analysis of metaphase chromosomes from invasively sampled cells which must then be cultured, taking 7-14 days for the final result (Ried, Landes, Dackowski, Klinger, & Ward, 1992). Although this method is able to detect a wide range of abnormalities, it is labour intensive and requires skilled analysts. The latency for prenatal aneuploidy results can become a source of anxiety and the burden for the mother and her family (Kowalcek, 2007). FISH can provide a result as rapidly as 24 hours, as it can perform analysis in uncultured interphase chromosomes. It is important to note that FISH should not be used as a standalone test, but as more of a rapid, initial diagnosis followed by karyotyping (Caine et al., 2005).
The applications of FISH can be extended to preimplantation genetic diagnosis, where fertilised embryos in vitro are tested for presence of genetic abnormalities and significantly increase pregnancy rates (Munne et al., 2010). One of the disadvantages of using FISH is that it can only detect approximately 80% of genetic abnormalities (Evans et al., 1999) as traditional FISH uses chromosome-specific probes (Evans et al., 1999). A new FISH technique, quickly replacing traditional FISH, is array comparative genomic hybridization (array CGH). This method allows for a genome wide detection by comparing the whole genome of test DNA with a standard DNA sequence and examines for any signal intensity differences. Signal intensity differences are indicative of deletion and duplications which superior than detecting a few chromosomal targets (Simpson, 2010).
FISH provides a rapid identification of microorganisms such as pathogenic blood stream infections or urinary tract infections (UTI), where it is vital to begin antimicrobial therapies at an early stage (Remco et al., 2006). Traditional diagnostic tools include using gram positive and negative stains, but FISH is able to provide information on spatial resolution and morphology, with sensitivity over 95% (Frickmann et al., 2017). Pathogenic blood stream infections can result in mortality so early treatment is critical. Alternative methods include PCR, but are generally more labour intensive. For diagnosis of UTI, it involves partial sequencing of 16s rRNA, which highly conserved amongst eukaryotes. However, the need to cultivate a bacterial sample would take 2-3 days, but with FISH it is possible to obtains results after 4 hours (Wu, Li, Wang, Pan, & Tang, 2010).
The basic principle that allows the use of FISH to characterise malignant tumours is that cancers are genetic disorders that involve high occurrences of chromosomal alterations. A normal cell would contain two copies of each chromosome which would appear as two copies of a probe in FISH. However, if there exists an abundance of cells that show aneuploidy, such as 3 copies of each chromosome (trisomy) or 4 copies of chromosome (tetrasomy), the tissue can be characterised as malignant. The following are a few examples where FISH has been widely used to diagnose cancers or is a potential technique.
25-30% of breast cancer patients experience a Her2/neu gene amplification, located on chromosome 17. Assessment of Her2 gene amplification by FISH is considered the gold standard (Tchrakian, Flanagan, Harford, Gannon, & Quinn, 2016) and was FDA approved in 2001 (Perez, Cortes, Gonzalez-Angulo, & Bartlett, 2014). Modern FISH techniques uses a dual-probe to assess gene amplification by considering gene copy number as well as Her2 to CEP17 ratio, where CEP labels are centromere for chromosome 17 acting as an internal control for comparison (Wolf, 2014). Another common test for Her2 gene amplification is using immunohistochemistry (IHC), but FISH is held in higher regard as it has a higher accuracy (96-97% vs 90-97%), greater reproducibility as well as a higher sensitivity and specificity (Pauletti et al., 2000; Press et al., 2002).
However, the downside of using FISH is that it is more costly and timely than IHC (Hicks & Tubbs, 2005). To overcome the cost barrier, FISH is currently used to clarify ambiguous IHC results. To address the time disadvantage, recent developments in Her2 testing include the automation of tests (Tchrakian et al., 2016). This allows high throughput testing and avoids inter-observer variability from manually counting dots in an image, improving both efficiency and consistency (Yoon, Do, & Cho, 2014).
Skin cancers or melanocytic neoplasms are routinely diagnosed using histopathological examination. Even though it is considered the ‘gold standard’ classification of a melanoma as benign or malignant has been reported to vary up to 25% (Lodha, Saggar, Celebi, & Silvers, 2008). These high discordance rates pose legal and patient risks, arising from the fact that some melanomas are morphologically ambiguous. This calls for objective genetic testing, where FISH has a high rates of sensitivity (87%) and specificity (96%) for melanocytic neoplasms (Gerami et al., 2009).
Recently FISH has been increasing in popularity to be use for urothelial carcinoma (UC), the most common type of bladder cancer. Traditional diagnosis involves urine cytology, which has a very poor sensitivity (26%), whilst FISH has much higher sensitivity of 95% (Sarosdy et al., 2006). For UC, FISH probes detect aneuploidy in chromosomes 3, 7, 21 and 9p21 band (Halling & Kipp, 2008). FISH is also especially useful for UC as it is more sensitive to all grades of UC. It is even able to detect UC months earlier than conventional cytology, as chromosomal alterations become apparent before morphological changes (Jones, 2006). Similar to melanoma, FISH has been found to be a valuable ancillary tool to clarify inconclusive findings (Gudjónsson et al., 2008).
A recent technological advancement is using a lab-on-chip device, where FISH can be performed on a small computer chip, millimetres wide, while dealing with extremely small fluid volumes. The future development of this technology will allow for high throughput samples diagnosis of various tumours (Lim et al., 2012).
Thus FISH is not only widely used cytological tool, it is able to provide accurate information efficiently and rapidly. Recent advancements in FISH includes array CGH, FISH automation and lab-on-a chip device, and future directions will foresee the implementation of these advancements in a clinical setting. The value of FISH is often found its use as an ancillary tool, as its able to give valuable insight into certain cell characteristics.
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