FISH Results and Analysis- A Complete Guide

“FISH results are analyzed using fluorescent microscopy. By looking into the fluorescent probe signals chromosomal abnormality can be investigated.” 

FISH—Fluorescence In Situ Hybridization is categorized into a molecular cytogenetic technique. Scientists use FISH to identify and study chromosomal alterations, particularly structural alterations. 

For instance, Translocations are difficult to study using conventional karyotyping, using FISH not only translocation but also deletion, duplication and other structural and numerical abnormalities associated with chromosomes can be investigated. 

Due to this reason, it is widely used in cancer studies and diagnosis. In this article, I will explain the process of studying and interpreting the FISH results. 

Everything that we are discussing in this article is explained in layman’s and simple language. One with a basic knowledge of genetics can easily understand it. 

Stay tuned! 

Disclaimer: The content presented herein has been compiled from reputable, peer-reviewed sources and is presented in an easy to understand manner for better comprehension. A complete list of sources is provided after the article for reference.

What are FISH results? 

FISH results are fluorescence signals obtained from the hybridization process. Here we are using various types of probes as per the requirement. For instance, a locus-specific probe or a gene-specific probe. 

This probe will bind to the gene or locus on the chromosome, if the gene is deleted or translocated on some other chromosome, it will still give a hybridization signal.  

Various fluorescent colors used in FISH are red, green, blue, yellow or a combination of two different colors in case of gene fusion or translocation. Keep in mind that the FISH technique is used to investigate 

• Numerical chromosomal anomalies, for instance, down syndrome. 
• Structural chromosomal anomalies. 
• Special chromosomal rearrangements like translocations and deletion. 
• Gene fusions. 
• Anomalies on centromeres. 
• Anomalies on telomeres. 
• Chromosomal or centromeric instability. 

Instrumentation requirements: 

Compound or routine microscopy can’t be used to investigate FISH results. We need a fluorescence microscopy system equipped with appropriate fluorescence filters. 

Filter should be used as per the dye used during the process. Take the necessary training to handle and use the fluorescent microscope. 

In addition, prepare and fix a slide as per standard slide preparation guidelines. After that, 50 to 200 interphase nuclei are investigated for statistical reliability. However, the present criteria may also differ from sample to sample as well. 

Keep in mind that you should investigate only good-quality nuclei. No quality measure parameters are available, experience helps to identify good quality fields for investigations. 

Now, it’s time to understand the results.  

How to study FISH results? 

We will divide this section into two different sub-sections. We first discuss the classic application of FISH– to investigate structural chromosomal anomalies.  

(In our online course on FISH, I explained all the results with examples in detail. Check out the course now!)

Structural chromosomal anomalies by FISH: 

I will try to explain the results in simple language so that you can understand. Here, we will have to spot the fluorescent signals as a colored dot in the field (for interphase nuclei). 

The number of spots may vary based on the experiment. For instance, if we use a single color probe for a single locus then certainly we will observe two same color spots. Check out the image below. 

Chromosomes are present in pairs, and so are the alleles or genes. Hence, for a single locus, we observe two color spots in the normal sample. For example, if we take an example of a HER2 gene, and use red fluorescence, we observe two independent red color spots on chromosome 17. 

Similarly, if we use two color FISH for two different genes, we will observe four different color spots. 

For example, a green fluorescent probe is used for the BCR gene located on chromosome 22 while the red color probe is used for the ABL gene located on chromosome 9. 

We normally observe two green and two red color spots in the nucleic field. However, in Acute Myeloblastic Leukemia, we observe the fusion of BCR-ABL genes due to the translocation between chromosomes 22 and 9. 

In this case, we observe only a single red and green spot and a fusion between both as a yellow or red-green fusion color. So the results should be something like this: 

Likewise, the fluorescence signal pattern varies for different experiments and probe types. We will explain about different types of FISH probes and their results in a separate article. 

Deletion: 

A deletion probe is used to investigate chromosomal deletions. Here, in the case of a dual probe, the signal pattern shows only a single color for the deleted locus. Check out the image below. 

Dual color break-apart probe: 

Now this situation is a bit tricky and needs expert observation to understand and investigate. Let’s try to understand it. The dual or tri-color break-apart probes are used to study translocations of the adjacent genes. 

In the image below, you can see that in the normal condition, both the adjacent genes are located on a pair of chromosomes, however, in the abnormal condition, one gene is deleted and translocated to another chromosome, and results in separate signals. 

Tricolor Break-apart probe: 

A similar principle for the dual-color break-apart probes is applied here in the tricolor break-apart probes. Instead of two, three different adjacent genes from a pair of chromosomes are investigated. Translocations and inversions result in aberrant interphase results. 

Fusion gene probe: 

We already explained dual fusion or fusion gene probe with an example of BCA-ABL gene fusion. Here, translocation results in fusion between two genes from non-homologous chromosomes and results in fusion probe signals. 

Gene amplification probes: 

Again a tricky situation. Here is what happens, a gene multiplies or expands abnormally and results in many fluorescent signals for a single gene. Many copies of a gene are observed in an aberrant interphase. 

Now, you get some hints, I guess! 

Moving further. 

Common FISH patterns for various types of structural chromosomal anomalies are discussed here. 

Numerical chromosomal abnormality by FISH 

Numerical chromosomal analysis is an easy task compared to structural anomaly analysis. So we will follow a similar pattern, and use a single color probe to determine the condition. 

For instance, for trisomy we observe three different single color spots, for monosomy we observe a single and for disomy, we observe two different single color spots in the field. 

Check out this table to know more. 

Notedly, sometimes two different probes can be used in a single experiment to investigate complex conditions. For instance, separate probes for trisomy 21 and 18 are used to investigate both conditions in a single experiment.

Wrapping up: 

FISH results are easy to investigate compared to conventional karyotyping. However, it also has technical limitations. False and non-specific signals are the common problem here. 

In addition, multi-color FISH further needs additional expertise and comprehensive experience to investigate the results. I hope you like this article. In the next article we will understand various types of probes,  their FISH results and why we use them. 

If you’re passionate about FISH and eager to build industry-ready skills, enroll in our course to kickstart your journey! Link is given here.

>> Basics to Advanced FISH.

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