FISH

Question 1

Which of the following counterstain dyes should be used to counterstain the cells with
Spectrum Green /Spectrum Red dual color signals?

Answer 1

DAPI(+)
Propidium iodine
Ethidium bromide
Acridine orange

Question 2

DAPI is a _________ counterstain.

Answer 2

green
yellow
blue(+)
red

Question 3

The process of fluorescence in situ hybridization is used to:

Answer 3

Structure a complete karyotype.
Show the repair of DNA sequences after
denaturation.
Visualize specific sequence of DNA with a
fluorochrome.(+)
Label colonies of cultured cells.

Question 4

Which of the following is critical to the success of a FISH procedure?

Answer 4

Probe can only be labeled with the fluorochrome.
Temperature of the denature solution.(+)
Slides should be aged at 60oC for at least overnight.
The FISH signal should be detected immediately after the hybridization.

Question 5

Formamide is used in the FISH procedure for which of the following purposes:

Answer 5

To be used to rinse off excess salt from the slides.
To modify a DNA probe so it can be detected by a
fluorochrome.
To artificially age the slide.
To lower the DNA melting point.(+)

Question 6

The purpose of the post hybridization wash is to:

Answer 6

Enhance the binding of the fluorochrome
Serve as a pretreatment for probe labeling
Remove unbound probe from the slide(+)
All of the above

Question 7

Which of the following is true about slide preparation for FISH?

Answer 7

Slides should be kept at room conditions.(+)
Slides should be treated overnight in a 90oC oven.
Slides should be prepared 1 week in advance and
stored in the refrigerator.
Slides should be protected from ultraviolet light

Question 8

When analyzing a sample stained with a FISH probe, the technologist notices a high amount of nonspecific cross-hybridization. Which of the following is the most likely cause?

Answer 8

The counterstain was not added.
The wash stringency was too low.(+)
The manufacturer produced a poor probe set.
The denaturation process did not work.

Question 9

When using the Applied Imaging system to capture the FISH image, which program should be chosen to use?

Answer 9

Fluorescent
Probe(+)
RXFISH
MFISH

Question 10

Which of the following statements is true about directly labeled probes?

Answer 10

Should be counterstained only with DAPI.
Require amplification of the probe signal.
Exposure to light should be avoided.(+)
Denaturation of the probe is not necessary.

Question 11

All of the following are advantages of array-CGH EXCEPT:

Answer 11

Genome-wide screening.
Use metaphase chromosomes(+)
Use DNA extracted.
Easy to distinguish gains from losses.

Question 12

Which of the following statement regarding test resolution is correct?

Answer 12

G-band karyotyping is a whole-genome approach with
resolution ranging from 100-500MB.
FISH is a whole-genome approach with resolution
ranging from 5-500 KB.
a-CGH is a whole-genome approach with lower
resolution.
BAC array has lower resolution than Oligo array.(+)

Question 13

Which of the following is/are the quality control measurement(s) for array-CGH?

Answer 13

Triplicate clones used.
Dye swap.
Equal amount of sample and reference DNA.
All of above.(+)

Question 14

Which of the following statements regarding array-based CGH is FALSE?

Answer 14

aCGH compares ratio of test DNA to normal reference DNA to identify copy number abnormalities across the entire genome in one assay
array CGH analysis creates the opportunity to transition from subjective to quantitative
results.
molecular technique that detects balanced genomic rearrangements (translocations and insertions) by comparing test DNA to normal reference DNA.(+)
facilitates genome-wide analysis of samples from which chromosome preparations are difficult or impossible.

Question 15

Which of the following is NOT an indication of aCGH test?

Answer 15

a 3 month-old baby boy with dysmorphic features.
a mother carries an inversion 9 by G-band karyotyping analysis(+)
a mother who has a history of 3 unexplained miscarriages.
a 2 month-old baby girl with multiple congenital abnormalities.

Question 16

All of the following are advantages of array-CGH EXCEPT:

Answer 16

Genome-wide screening
Use metaphase chromosomes(+)
Use DNA extracted.
Easy to distinguish gains from loses

Question 17

FISH

Answer 17

Fluorescent in situ hybridization (FISH) is a technique that hybridizes fluorescent probes onto cytological slide preparations. This hybridization allows visualization of target DNA sequences that are of clinical interest.

Question 18

FISH basic requirement

Answer 18

is the presence of undegraded DNA

Question 19

FISH can be performed on the following:

Answer 19

chromosome preparations
touch preparations made from tumor specimens
cytospin preparations
paraffin-embedded tissue
smears (either blood or bone marrow)
disaggregated nuclei from tissues (processed by cytospin or smears)
uncultured amniocytes
blastomere/blastocyst for preimplantation analysis

Question 20

Probe

Answer 20

A relatively small piece of DNA that is used to find another piece of DNA (target.) Targets can be genes, section of chromosomes or whole chromosomes.

Question 21

Paint

Answer 21

Painting probes, also called whole chromosome paints (WCPs) hybridize to the unique sequences which cover the length of an entire chromosomes or chromosome arms. These probes are useful in studying marker chromosomes, translocations and aneuploidy in metaphase cells. These probes are not useful for interphase analysis. This is because the chromosome domain is dispersed in the interphase part of the cell cycle.

Question 22

Centromere probe

Answer 22

Centromere probes hybridize to alpha-satellite DNA. Alpha satellite DNA is repetitive DNA sequences located at the centromere of each chromosome. This type of DNA is composed of a 171 base pair repeat that is polymorphic from individual to individual. Most alpha satellite centromeric probes give a large, bright signal. These probes are useful in determining aneuploidy of specific chromosomes in both metaphase and interphase cells.

Question 23

Other Satellite Probes

Answer 23

Like the alpha satellite probes these probes can be used on both metaphase and interphase cells.
Beta satellite probes - Beta satellite DNA is composed of multiple 68 base pair repeats located on the short arm of the acrocentric chromosomes.
Classical satellite probes - Classical satellite DNA is located in the heterochromatic regions of chromosomes 1, 9, 16, and Y. This DNA is highly polymorphic and composed of numerous tandem repeats of the AATGG sequence.
Telomere probes - Telomere DNA is located at the terminal ends of the p and q arms of each chromosome. This DNA is composed of tandem repeats of the TTAGGG sequence. Non-specific telomeric FISH probes target this region. The centromeric end of TTAGGG repeats is the telomere associated repeats. Unique subtelomeric probes target unique DNA sequence immediately proximal to the telomere associated repeats region and are now available for each chromosome arm.

Question 24

Fusion Products

Answer 24

Dual-color/Single fusion
Translocation probes will show fusion products in abnormal cells. The critical regions of the two genes involved in the translocation are labeled either red or green. When the red and green dyes come in close proximity of each other, a flare will occur which will appear white or yellow in color. Normal cells will have four signals (two red and two green). Abnormal cells will have three signals (one red, one green and one fusion). These probes are useful in determining translocations in both metaphase and interphase cells. The positive signal pattern for this probe is 1F1R1G. The cut-off value for a positive result is typically high due to the decreased sensitivity and specificity of this probe due to random colocalization. Colocalization occurs when two or more signals are very close spatially causing the signal to appear as one as opposed to two or more separate signals. Single fusion is not a reliable measure of minimal residual disease (MRD).
Dual-Color/Extra Signal
The critical regions of the two genes involved in the translocation are labeled either red or green. This probe uses DNA sequences which flank the breakpoints on one of the chromosomes. The positive signal pattern reveals a single fusion signal and extra signal. The observed signal pattern for a translocation using this probe is 1F2R1G where the smaller, extra single-colored signal is from one of the translocated chromosome. The specificity of this probe is typically high when an extra signal is observed.
Dual-Color/Dual Fusion
The critical regions of the two genes involved in the translocation are labeled either red or green. The positive signal pattern reveals double fusion signals. Generally, a dual color, dual fusion probe will span larger DNA areas along both chromosomes involved in the translocation, thereby, increasing the sensitivity and specificity of this probe type. The observed signal pattern for a translocation is 2F1R1G. This probe has the highest sensitivity and specificity for detection of translocations, and is very useful for monitoring MRD.
Dual-Color/Break-Apart Rearrangement
The critical region of the gene is covered by two different color probes flanking the locus involved in the rearrangement or translocation. A normal configuration is denoted by a yellow signal pattern (2F). In the event of a rearrangement or translocation, the signal pattern is 1F1R1G. The specificity of this probe is typically high when break-apart signals are observed.

Question 25

Locus-specific probes

Answer 25

Probes in this group identify a specific gene or locus on a chromosome and hybridize to the target DNA at that location. These probes hybridize to unique sequences in the genome. These sequences code for important genes that play an important role in human disease management. Examples are the her-2neu gene that is amplified in breast cancer patients and the C-MYC gene that is amplified in patients with leukemia.

Question 26

1. Pretreat Slides
   - interphase analysis
   - metaphase analysis

Answer 26

Chromosome slides prepared with the standard hypotonic and acid /methanol washes are easily used for the FISH procedure. It is important that the metaphases and interphases are free of cytoplasm and debris as this can interfere with the hybridization of the probe and can cause an increase in background signal. (If slides continue to have an excess of cytoplasmic background, pretreat slides with an acid wash or proteinase K treatment to remove cytoplasmic proteins.)
Non-chromosome preparations (smear, cytospins) should be placed in a coplin jar of 1:1 glacial acetic acid and methanol for two minutes. This treatment will lyse any red blood cells that may be on the slides. Following this, place the slides in a fresh coplin jar of 1:1 glacial acetic acid and methanol for five minutes.
metaphase analysis (cont.)
Routinely slides for FISH should be stored at room temperature in a dust-free slide box.
Slides less than two weeks old must be artificially aged. Treating slides in a coplin jar of 2xSSC (pH 7.0) @ 37oC for 30 minutes does this. Following this pretreatment, slides should be dehydrated in 70%, 85% and 100% ethanol for two minutes each at room temperature.
Slides more than two weeks old need not be treated with 2XSSC prior to denaturation.
Slide storage - FISH slides may be stored at -20oC for up to six months if they can not be processed right away. They can be stored at -80oC for up to one year. These slides when thawed should be pretreated with 2xSSC before denaturation.

Question 27

2.Denature and Dehydrate Slides

Answer 27

In order for a single-stranded probe to hybridize to the target DNA of the specimen, the target DNA must be denatured. Denaturing breaks the hydrogen bonds that hold the complementary strands of DNA together. Once these bonds are broken, double-stranded DNA becomes single-stranded DNA. After denaturation, a single-stranded probe can hybridize to complementary sequence in the genome.
Slides should be denatured at 72oC for 2 minutes in 70% Formamide/2xSSC (pH 7.0). The pH of the formamide is critical as well as the temperature. If more than two slides are denatured in the same coplin jar at one time, a degree in temperature should be added for each additional slide. (Example: Denature at 74oC for 4 slides). Formamide lowers the melting point of DNA. An aqueous solution of DNA would have to be heated close to boiling before the strands will separate. When the strands of DNA separate, DNA is said to melt, or become denatured.

NOTE: If slides have been aged more than one month at room temperature, the denaturation time should be increased up to 5 minutes.

Immediately following denaturation, slides should be dehydrated in cold ethanol. Rapid chilling of DNA that was denatured by heating locks the DNA in the denatured state. Treat slides for 2 minutes each in 4oC coplin jars of 70%, 85% and 100% EtOH. After dehydration, allow the slides to air dry.

Question 28

3.Denature probe

Answer 28

Probes containing repetitive sequences can self-hybridize in the tube. Because of this phenomenon, satellite and painting probes must also be denatured at 72oC for 5 minutes before use. Denatured probes should be placed on ice until ready for use. (NOTE: painting probes should be placed at 37oC for 30 minutes to one hour after denaturation before being used).

Question 29

Co-Denaturation

Answer 29

Instead of denaturing slide and probe separately, co-denaturation becomes more popular and efficient with the availability of Hybrite. Probes are added to slide directly before denaturation. Slide is coverslipped, sealed with rubber cement and placed in Hybrite for co-denaturation and hybridization.

Question 30

4.Hybridize Slides

Answer 30

Following denaturation the following slide preparation steps should be followed.

10ul of probe should be added to each slide.
The probe area should be covered with a 22 x 22 coverslip.
The edges of the coverslip should be sealed with rubber cement.
The slide(s) should be placed in a humidified chamber at 37oC for 30 minutes to overnight depending on probe used.
Repetitive probes hybridize much faster than painting or unique sequence probes.
Following hybridization, slides must be washed in order to remove unbound probe and probe that has cross-hybridized to nonspecific regions of DNA. Slides are washed for 2 minutes at 72oC in various concentrations of SSC (0.25 - 2X SSC). The concentration of SSC selected depends on the probe that was hybridized. For example, a unique sequence probe should be washed in 2X SSC. An alpha-satellite probe that contains repetitive DNA sequences should be washed in 0.25XSSC. The lower the salt concentration the more effectively the probe is washed off of the slide. This is described by the term stringency.

Question 31

Direct-labeled slides

Answer 31

Direct-labeled probes have a fluorochrome tagged nucleotide incorporated into the DNA sequence. This process uses either nick translation, PCR, or random DNA priming techniques. These slides can be viewed under a fluorescent microscope directly after hybridization and wash steps without detection.

Question 32

Indirect-labeled slides

Answer 32

Indirect-labeled probes are tagged with a hapten such as biotin or digoxigenin. These haptens can be detected with avidin or antidigoxigenin antibodies with conjugated fluorochromes. This results in the probe being tagged with multiple fluorochrome molecules at each nucleotide with a hapten molecule.

Question 33

5.Use an Appropriate Counterstain

Answer 33

In order to visualize the entire metaphase or interphase cell the FISH probe must be counterstained with an appropriate fluorochrome. Typically, probes labeled with Fluorescein isothiocyanate (FITC, green) are counterstained with Propidium iodide (red)
Probes labeled with either Rodamine (red) or Texas Red (dark red) are counterstained with DAPI (blue)
Slides hybridized with more than one probe (red and green) are counterstained with DAPI (blue)

Question 34

6.Select Correct Filter With Fluorescence Microscopy

Answer 34

The filter that is used to visualize FISH slides is chosen based on the fluorochrome(s) that were used. Fluorescence microscope filters consist of three individual filters: the excitation, barrier and emission filter. For single probe FISH a dual bandpass filter can be used to view both the probe and the counterstain. For dual probe FISH, a triple band pass filter is used. These filter sets allow only certain wavelengths of light to be visualized.

Question 35

7.Score Appropriate Type and Number of Cells

Answer 35

Interphase - Two hundred to five hundred interphase cells should be scored. The interphase cells selected should be nonoverlapping with other cells, have distinct nuclear borders, and show no obvious cross-hybridization or background signal.
Metaphase - Ten to fifty metaphases should be scored. The number of metaphases analyzed depends on the probe and its application. For example, congenital disorders require the analysis of fewer cells. Mosaic conditions such as occur with chromosomal aneuploidy seen in prenatal clinics or clonal abnormalities observed in cancer clinics require analysis of more cells to rule out mosaicism and to establish clonality. Analysis of 30 cells will rule out a mosaicism of 10 percent with a confidence of 95%. Counting 50 cells will rule out a mosaicism of 6% with a confidence of 95%.

Question 36

8.Capture cells or representative field with imaging system or photographs

Answer 36

It is important to capture images for the patient file that accurately reflect cells analyzed under the microscope. Images should show nuclei or metaphases free of distortion with clear, bright and discrete signals without nonspecific background. Nuclei should not be odd-shaped or scrapped. In addition, nuclear borders should be clearly identifiable and not overlapped.

Question 37

Perform quality control

9.Use of control probes

Answer 37

is essential in reducing the number of false positive and false negative results.
Examples:
Chromosome enumeration - Two centromere probes should be hybridized to two different chromosomes. Each probe should be labeled with a different colored fluorochrome.
Microdeletions - Two unique sequence probes located on the same chromosome are used to detect microdeletions. One probe lies within the critical region and one probe lies outside the critical region.
Gene amplification - One centromere probe labeled with one fluorochrome and an oncogene probe labeled with another color fluorochrome are used to detect copy number changes in oncogenes that are associated with neoplastic transformation.

Question 38

10. Use of Control Specimens

Answer 38

A positive and a negative control slide should be processed with each hybridization experiment and with each new lot of probe. The use of control slides validates abnormal results found in single probe hybridizations.

Question 39

11. Establishment of Acceptable Ranges

Answer 39

When a probe is used in a laboratory for the first time a minimum of 10 normal, peripheral blood control slides obtained from ten different specimens should be assayed in order to determine acceptable ranges. Two or more independent scorers should read these slides. Their results should be within 5% of each other.

Question 40

Correlating Results with Standard Cytogenetics

Answer 40

Perform FISH on the same sample that has been studied with G-banding
FISHing G-banded slides
Inverted DAPI

Question 41

Use of Appropriate Recording Protocol

-Correlation with Standard Chromosome Analysis

Answer 41

The majority of FISH probes are used as an adjunct test. Standard chromosome analysis should be performed on the sample specimen that is studied by FISH. Two ways to combine both analyses is to karyotype inverted DAPI images using a computer imaging system or to use the sequential staining technique of performing FISH on G-banded slides.

Question 42

-Sequential G-Bands to FISH

Answer 42

Locate and photograph G-banded cells on the slide.
De-oil slides by soaking in xylene or a xylene substitute for one minute.
Rinse slide twice in fresh 3:1 methanol:acetic acid fixative for five minutes each. (note: very important to obtain a clean slide or denaturation/hybridization will not be successful)
Dry slide. Incubate in 2XSSC at 37oC for 30 minutes; Codenature slide and probe for two minutes, then incubate at 37oC overnight in a humidified chamber.

Question 43

Use of Appropriate Recording Protocol

-Statement FDA Approval Pending

Answer 43

FDA cleared - indicates that this probe can be used as an adjunct to standard karyotyping.

FDA approved - indicates that this probe can be used as a stand alone test.

Vysis’ FDA Cleared FISH Kits

CEP® 8 SpectrumOrange DNA Probe Kit
CEP 12 SpectrumOrange DNA Probe Kit
CEP X/Y Dual Color DNA Probe Kit
AneuVysion Assay (chrs. 13, 18, 21, X &Y & DAPI)
Urovysion (Aug, 2001)
Vysis’ FDA Approved FISH Kits

LSI HER-2 DNA Probe Kit (PathVysion) Spring 2001

Question 44

Troubleshoot

1.Unacceptable/Unanalyzable Specimens

Answer 44

Specificity - The percent of signals that hybridize to the correct loci.
Sensitivity - The percent of cells with the expected signal pattern.
Unacceptable/unanalyzable specimens will not meet the following three criteria.
Probe intensity - The probe signal should be bright and easily detected.
Cross hybridization - Metaphase spreads should be analyzed to determine target specificity. Nonspecific binding of the probe to non-target areas could indicate improper stringency during the FISH procedure.
Background - High levels of background probe signal (not occurring within nuclear borders) indicates improper wash conditions and will lead to an increase in false positives. To correct this problem the slide should be rewashed with a lower concentration of SSC to increase stringency. This procedure is needed to remove nonspecific probe.
Soak off coverslip in 1XPBD (or other detergent wash).
Rinse slide in fresh 1XPBD for 2 minutes.
Incubate slide in appropriate SSC solution for 5 min at 72oC.
Repeat detection and counterstain steps.

Question 45

Troubleshoot

2. Inadequate Hybridization

Answer 45

Inadequate hybridization often occurs because of factors that affect stringency. Fewer than 2% of cells should show an absence of probe signal to be acceptable.
The following are conditions that can cause inadequate hybridization:
Improper denaturation of either the target DNA or the probe due to improper temperature, pH of denaturing solution, or too much cytoplasm or unclean slides.
Too high of stringency during the post hybridization wash procedure due to high temperature or too low salt concentration used.
Hybridization conditions were not appropriate. Temperature of incubator, coverslip not mounted properly (air bubbles) or inadequate hybridization time used.
Microscope not properly working or set up with inappropriate filters.