Fluorescence In Situ Hybridization

Fluorescence in situ hybridization essentially indicates the fluorescent reporting of ISH. A sketch of routine FISH protocols for embedded tissue sections and cell preparations shown in Table 2 highlights its origin in the common ISH platform. FISH is commonly identified with its most popular application in cytogenetics, in contrast to the general notion of ISH as a RNA- detecting technique. In recent years, the growing commercialization of FISH technology has guided its way into the disease management armamentarium in a clinical laboratory. Although, the basic methodological considerations are carried over from ISH described earlier, several points of distinction deserve further description.

4.1. CELL CYCLE PHASE OF THE TARGET CELL As mentioned earlier, the major revolution in cytogenetics brought in by FISH was the possibility of cytogenetic analysis in any phase of the cell cycle, unlike the metaphase requirement of classical cytogenetic analysis by chromosomal banding. It was Cremer who coined the term "interphase cytogenetics" (17,18). The metaphase analysis requires in vitro cell culture. Thus, it is time-consuming as well as labor-intensive and is often futile in certain tissue types. Surpassing this requirement in an interphase analysis not only offers a unique ease with time and effort but also makes it applicable to cytological and archived histological specimens equally well. The interphase analysis also allows for a simultaneous phenotype assessment. For instance, Rigolin et al. (34) showed that the monocyte-derived dendritic cells in patients with myelodysplastic syndromes carry the clonal cytogenetic abnormality. The same group later also showed a multilineage involvement in a 5q- syndrome using interphase FISH (33). The metaphase analysis requiring dividing cells relies largely for accuracy on the proliferative potential of target cells, which varies significantly among different genotypic and phenotypic clones in a given clinical specimen. Thus, an inevitable preferential enrichment of rapidly proliferating clonal populations determine the eventual metaphase cytogenetics readings. This is obvious especially in the analysis of bone marrow disorders. The interphase analysis surmounting this difficulty could account for even minor cytogenetically abnormal clones (36). The major limitation of interphase cytogenetics is the detection of only a specific abnormality and not permitting the localization of hitherto unidentified cytogenetic abnormality. Metaphase analysis scores

Table 2 General FISH Protocol

Steps involved in FISH

Paraffin-embedded tissue sections

Cell preparations and isolated nuclei

Intended application Fixatives

Aging

Pretreatment

Protease treatment

Postfixation Denaturation

Hybridization

Posthybridization wash

Detection (necessary only for indirect FISH)

Counterstain/mounting Visualization

Interphase cytogenetics

4-10% Formalin

None

(a) Incubation at 52°C overnight

(b) Deparaffinization using xylene or commercial agents like HemoDe and graded alcohol

(c) Incubate sections in 1 M sodium thiocyanate (NaSCN) at 80°C for 20-30 min

0.05% (wt/vol) pepsin in 0.01N HCl, pH 2.0, at 37°C for 10-20 min

4% Buffered formaldehyde

Target: 70% formamide in 2X SSC, pH 7.0 at 72°C-74°C for 1-2 min

Probe: 50-60% formamide in 2X SSC, pH 7.0 at 72°C-74°C for 1-2 min

Target incubation with probe prepared in a 2 X SSC solution containing 50-60% formamide and 10% dextran sulfate, pH 7.0 at 37°C-42°C overnight

(1) Incubation with fluorescent labeled antibiotin or antidigoxigenin antibody

(2) Alternative amplification with ezyme-Fluorophore- tyramide system

DAPI with antifade

Epifluorescence microscopy with appropriate filters

Interphase or metaphase cytogenetics Carnoy's fixative (methanol: acetic acid :: 3:1)

or 4% formalin Overnight or 2X SSC solution at 73°C for 2 min None

0.05% (wt/vol) pepsin in 0.01 N HCl, pH 2.0, at 37°C for 10-20 min

1% Buffered formaldehyde

Target: 70% formamide in 2X SSC, pH 7.0 at 72°C-74°C for 1-2 min

Probe: 50-60% formamide in 2X SSC, pH 7.0 at 72°C-74°C for 1-2 min

Target incubation with probe prepared in a 2X SSC solution containing 50-60% formamide and 10% dextran sulfate, pH 7.0 at 37°C-42°C overnight

(1) Incubation with fluorescent labeled antibiotin or anti-digoxigenin antibody

(2) Alternative amplification with ezyme-fluorophore- tyramide system

DAPI with antifade

Epifluorescence microscopy with appropriate filters

Source: refs. 32 and 33.

merit in this respect (for review, see ref.32). On the other hand, submicroscopic chromosomal rearrangements missed in banding analysis such as those known to occur in chronic myelogenous leukemia (CML) could be visualized by conducting FISH on metaphase and/or interphase cells (37).

4.2. DNA PROBES Generally, most commercially available DNA FISH probes are prepared by selecting clones of yeast, bacterial, or P1 artificial chromosomes (YACs, BACs, or PACs respectively). Some probes are also prepared by PCR amplification of DNA. As shown in Table 3, there are primarily three types of chromosomal DNA probes that target the centromeric region, the entire length of chromosome, or the specific region of a particular chromosome (38). The common application of the repetitive sequence centromeric probes for detecting numerical chromosomal abnormalities have earned them the name "chromosome enumeration probes" or CEP®s. The other two are named after their target range as the whole chromosome paints (WCP®) or locus-specific identifiers (LSI®), respectively. Additionally, owing to the importance of subtelomeric and telomeric regions in chromosomes in some cancers and congenital disorders, special LSI probes are available for these regions as well. Figure 2 shows photomicrographs illustrating different chromosome region targets of FISH probes detailed earlier. Further, the design of LSI probes targeting specific known balanced translocations has undergone considerable evolution in the last decade, minimizing the false-positive and/or false-negative rates. This is most apparent in the design of probes for t(9;22) translocation that results in a pathognomonic fusion BCR-ABL gene in CML (39). The shift in the probe design for LSI BCR-ABL from independent fluorophore-labeled probes targeting only one side of the break point on the two chromosomes, resulting in a single fusion signal to the probes spanning both sides of the breakpoints on both chromosomes and resulting in two fusion signals has remarkably improved the specificity and considerably reduced the false-positive rate to <1% (37). To assess the rearrangement of a gene with several translocation partners, the LSI design (break-apart probe) could include a combination of two probes labeled with distinct fluorophores and specific for sequences 5' or 3' of the breakpoint region within the rearranging gene. The separation of the two colors would thus be indicative of a rearrangement. Figure 2d,e shows examples of two unique LSI probe designs. When determining gene copy number, it is utmost important to have a built-in hybridization control. Usually, a centromeric or telomeric probe specific for the chromosome bearing the gene of interest is best suited as a built-in control. This is not only true for a gene deletion but is often needed in accurately determining the gene amplification status. In breast cancer, polysomy 17-associated increase in the HER2/neu gene copy number estimated using a combination of the HER2/neu LSI probe and a built-in CEP 17

Table 3

Comparison of Different Probe Designs

FISH probe

Target Cell type

Use of tissue sections Major application

Chances of probe cross-hybridization

Pericentromeric alpha (and/or beta) DNA repeats

Metaphase or interphase

Possible

(a) Assessment of numerical chromosomal abnormalities

(b) As built-in controls for LSI gene detecting deletion and amplification

Possible under less stringent hybridization conditions

Specific DNA locus

Metaphase or interphase

Possible

(a) Assessment of known chromosomal structural abnormalities

(b) Detect locus specific abnormalities

Entire length of individual chromosome

Metaphase only

Not Possible

(a) Identification of structural abnormalities on a specific chromosome(s) of suspect

(b) Identification of the origin(s) of a marker chromosome in a M-FISH assay

Negligible control has been reported to relate in all likelihood to a normal protein expression pattern. Although a true amplification of the HER2/neu amplicon on chromosome 17, which is seen as a multifold increase over the chromosome copy number, results in abnormally high levels of HER2/neu protein and, in turn, has significant therapeutic implications.(40,41) Additionally, such built-in controls provide a parameter for determining nuclear truncation, especially in tissue sections, where only the nuclei with at least one of each signal type should be included in the final analysis.

In contrast to mRNA ISH described earlier, the cytogenetics FISH probes, which are mostly double stranded, require denat-uration of probe and target prior to the hybridization step (Table 2). As emphasized by van Stedum and King (32), the use of blocking unlabeled human DNA (e.g., Cot-1 DNA) and for-mamide in a probe mixture during denaturation/hybridization is essential to suppress the hybridization of probe to nonspecific repetitive DNA sequences within the target. Formamide is known to control the melting point of duplex nucleic acids (32). The automated temperature-controlled protocols are available for denaturation/hybridization with equipments like HYBrite™.

4.3. FISH SIGNAL ENUMERATION In contrast to the ease in discerning the positivity of mRNA ISH, the signal patterns in cytogenetic FISH assays could be challenging and might need practice for perfection. Especially with LSI signals, the knowledge of probe design and the understanding of the specimen are extremely helpful. The factors that influence the quality and clarity of cytogenetic FISH signals are the adequacy of the amount of target DNA in a specimen, pretreatment of the specimen, denaturation/hybridization conditions, and duration, to name a few. An elaborate account of troubleshooting tips and signal enumeration guide can be found in the review by van Stedum and King (32). A few salient points are as follows:

1. Excessive protease digestion or overdenaturation could damage morphology as well as cause extremely weak or even loss of signals. On the other hand, underdigestion with protease or other pretreatments could preserve morphology, but might compromise signal intensity.

2. Fluorescence in situ hybridization signals are very specific, relatively tiny, and might assume a round, oval, or diffuse shape depending on the chromosome target as well as the chromatin structure within that region.

3. A use of oil-immersion objective lens (x40, X60, or X100) and focusing up and down to view signals in all optical planes of the section are essential for accuracy.

4. Sometimes, a signal could appear split, which should be counted as one. In such cases, typically two very closely juxtaposed same fluorophore signals of approximately equal size are seen that are not separated by a distance larger than the diameter of either spot.

5. Overcrowded, superlayered, or inadequately counter-stained areas of the specimen should be discounted from enumeration.

6. Use of the appropriate excitation/emission filters is a basic prerequisite.

Table 4 addresses a few points of common skepticism surrounding the FISH technique.

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