Tentative paragraphs:

1. FISH (or DNA-FISH)
2. RNA-FISH

3. Immuno-FISH

4. Chromosome painting

(Rachele Rosso): Fluorescence in situ hybridization (FISH) is a kind of cytogenetic technique which uses fluorescent probes binding parts of the chromosome to show a high degree of sequence complementarity, and it  can be used to find out where the fluorescent probe bound to the chromosome. FISH is often used for finding specific features in DNA, and to detect and localize specific RNA targets (mRNA, lncRNA and miRNA).This technique provides a novel way for researchers to visualize and map the genetic material in an individual cell, including specific genes or portions of genes. Different from most other techniques used for chromosomes study, FISH has no need to be performed on cells that are actively dividing, which makes it a very versatile procedure. It is composed by 4 main passages:

• A probe complementary to the known sequence is made and it is labelled with a fluorescent marker, as for example fluorescein, by incorporating nucleotides that have the marker attached to them;
•  Chromosomes are put on a microscope slide and denatured;
• The probe is denatured and added to the microscope slide, allowing the probe hybridize to its complementary site;
• The excess probe is washed off and the chromosomes are observed under a fluorescent microscope. The probe will show as one or more fluorescent signals in the microscope, depending on how many sites it can hybridize to.

FISH is widely used for several diagnostic applications as for example identification of numerical and structural abnormalities, characterization of marker chromosomes, monitoring the effects of therapy, detection of minimal residual disease. Moreover it has many applications in research as gene mapping or identification of amplified genes. FISH is also used to compare the genomes of two biological species to deduce evolutionary relationships.

A techniqe which derives from FISh is  the multiplex in situ hybridization (M-FISH). It represents one of the most significant developments in molecular cytogenetics of the past decade; it is a 24-color karyotyping technique and is the method of choice for studying complex interchromosomal rearrangements. The process is composed by three main steps:

• The multiplex labeling of all chromosomes in the genome with finite numbers of spectrally distinct fluorophores such that each homologous pair of chromosomes is uniquely labelled;
•  The microscopic visualization and digital acquisition of each fluorophore using specific single band-pass filter sets and dedicated M-FISH software. These acquired images are then superimposed enabling individual chromosomes to be classified based on the fluor composition in accordance with the combinatorial labeling scheme of the M-FISH probe cocktail used;
• The detailed analysis of these digitally acquired and processed images to resolve structural and numerical abnormalities.

5. FISH: advantages and disadvantages

(Simone Rocco): The development of banding techniques in the 70’s led to a first revolution in cytogenetic field, allowing an accurate definition of chromosomes and their abnormalities. The karyotyping study has increased our knowledges in the onco-hematology field, becoming necessary for a correct diagnostic classification. The major limitation of conventional cytogenetics is that the cell must be in mitosis in order to be correctly analyzed. Moreover, despite the presence of high resolution methods, the conventional cytogenetic analysis is able to detect only chromosomal rearrangements that affect more than 3 megabases (Mb). All these problems have been resolved thanks to the introduction of the Flourescence in situ hybridization (FISH) at the end of the 80’s. This technique is based on the ability of a DNA probe, marked with a fluorophore, to bind specifically to a complementary DNA target sequence. FISH can be used for metaphasic chromosomes, interphase nuclei, chromatin fibers or DNA microarrays. An interesting point is that the FISH conducted on interphase cells (Interphase FISH or iFISH) was the method that produced the greatest results in onco-hematology diagnostics. The probes used in FISH can be different: the whole chromosome painting probes permitted us to indentify chromosomes involved in structural anomalies, while the single-locus probes were fundamental not only in gene mapping, but also in the detection of break points in chromosomal translocations.

The chronic lymphatic leukemia (CLL) is the best example to understand what was the contribution of iFISH in the study of cell populations with a mitotic index of less than 1%. The chromosome analysis of patients suffering from CLL often does not provide results for the absence of metaphase states or demonstrates a normal chromosomal kit in 40-50% of cases. Instead, iFISH identifies abnormalities of the karyotype in 65% of patients analyzed during the diagnosis or during a stable phase of the disease and in 88% of patients with a progressive disease (?). Another example is the acute B-cell lymphoblastic leukemia (ALL): 106 karyotype alterations were defined thanks to FISH, while only 34 with banding techniques.

However, we have to consider that this technique has its limits. In fact, we must know in advance what type on chromosomal anomaly we expect to find, in order to be able to choose the most appropriate probe for the analysis. Furthermore, since a limited number of fluorochromes can be used, the FISH is not able to analyze simultaneously more than three anomalies. However, also all these limits have been recently resolved thanks to the introduction of multiplex-FISH (M-FISH) and Spectral Karyotyping (SKY), which were previously discussed. In terms of using the FISH technology around the world, we found that the FISH technique has very limited use in developing countries because of unavailability and lack of expert knowledge.