Interphase FISH study experiments in chronic myeloid leukemia

Summary

The purpose of this chapter is to apply fluorescence in situ hybridization to the study of interphase nuclei in patients with chronic myeloid leukemia (CML). In our experience, FISH has been used to detect all forms of BCE/ABL fusion-forming Philadelphia (Ph) chromosomes FISH is a valuable adjunct to routine cytogenetic studies and can be applied to the same specimens. Because FISH can be used to quantify the mid-dividing phase of proliferating tumor cells and nonproliferating interphase cells, it is particularly useful for evaluating the efficacy of therapy using peripheral blood or bone marrow. This chapter discusses the ways in which FISH-related assays with BCR and ABL probes can be empirically obtained and aspects of confirming their validity. It also discusses ways to ensure the quality of FISH assays with BCR and ABL probes in routine clinical practice.

Operation method

An interstitial FISH study of chronic myeloid leukemia

Materials and Instruments

DAPI Ethanol Formamide Glacial Acetic Acid Hybridization Buffer Methanol Nonidet P40 SSC
Coverslip BCR ABL Dual Color Dual Fusion Probe

Move

I. Samples

1. Best results are obtained with cultured bone marrow and peripheral blood harvested according to standard cytogenetic procedures for hematologic malignancies.

2. For HSH studies, it is important to use freshly prepared slices. Cells fixed in methanol and ice acetic acid are stored at -7.0°C until needed for FISH studies before titrating the slices.

3. Peripheral blood and bone marrow smears can also be processed in this way.

4. pH chromosome-positive control slides with normal and abnormal cells should be included in the study of each batch of patient samples.

II. Specimen Preparation

1. Scan the slide with a phase contrast microscope and locate an area that includes a sufficient number of interphase cells. These cells should be well formed and there should be a minimum of overlapping cells. If this slide is not satisfactory, it is better to prepare another slide rather than continue with the following procedure.

2. Bake the newly prepared slide in an oven at 65°C for 1 h or in an oven at 90°C for 10 min. Do not prolong the baking time at high temperatures (60-90°C).

3. Place this slide in a staining vat with 2XSSC solution at 37°C for 1h.

4. Dehydrate the slide by placing it sequentially in 70%, 85% and 100% ethanol for lmin each at room temperature. Air dry.

Denaturation and hybridization Staining cylinder method

1. For intermediate schizophase studies, prepared slides are denatured in 70% formamide at 74°C for Imin; for interphase studies, the denaturation time is 2 min.

2. The slides were dehydrated by placing the prepared slides in 70%, 85% and 100% ethanol for 1 min each at room temperature. Air drying.

3. Take 3 juL of LSI?BC scale/ABL probe working solution into a 0.65 mL small centrifuge tube and centrifuge for 10s.

4. Float the probe mixture in a water bath at 74°C for 5 min to denature.

5. Remove the centrifuge tubes from the water bath and immediately add the probe mixture to the prepared slides and place the slides in a slide warmer at 45 °C.

6. Place a circular 12 mm coverslip over the hybridization area. Seal the slide with rubber cement. Place the prepared slides in a wet box at 37°C for 8?20 h.

7. elute after hybridization.

HYBrite (Vysis, Inc., DownersGrove, IL.) co-denaturation methods

1. 3 uL of LSI BCR/ABL probe workup was added directly to the prepared chromosome slides. Cover the hybridization area with a round 12 mm coverslip. Seal the coverslip with rubber cement.

2. Fill the HYBrite tank with water. Be careful not to overfill the trough with water as this can interfere with the DNA denaturation process.

3. Set up the HYBrite program as follows: melting temperature 80°C, melting time 5 min, hybridization temperature 37°C, hybridization time 20 h.

4. Place the slide in the HYBrite slot and let the machine perform the hybridization process.

5. Remove the slide from the HYBrite tank and perform the subsequent post-hybridization elution step in 3.4.

IV. Post-Hybridization Elution

1. Remove the playdough. Carefully remove the coverslip and discard.

2. Place the slide in a staining vat containing 0.4XSSC at 74°C for 2 min.

3. Remove the slide and place it in a staining vat containing 2XSSC/0.1% NP-40 for 1 min at room temperature.

4. Remove the slide from the staining vat with the edge of the slide touching a paper towel to remove the excess 2XSSC/0.1% NP-40.

5. Add 10ul of DAPI-I working solution to the hybridized area.

6. Place a coverslip over the hybridization zone. Gently press the coverslip to remove air bubbles. Prepare the slide for microscopic analysis.

V. Microscope

1. Use a high quality fluorescence microscope with a 100ul mercury lamp.

2. Use FITC and Texas Red dual-channel settings to observe signals simultaneously. Use FITC or Texas Red single-channel setup to observe a single fluorescence.

3. Use a computer with appropriate image analysis system software to capture images of cells to be recorded.

VI. Grading Criteria

1. The Vysis probe produces a green signal (G) and the ABL probe produces a red signal (R) with blue background chromatin. The observed BCR/ABL fusion signal is either a red and green contact signal or a yellow signal. Later in this chapter, F is used to refer to the BCR/ABL fusion signal.

2. Normal interphase or midphase cells have 2 red and 2 green signals and no F signal (Figure 2).

3. Abnormal nuclei with one Ph chromosome have either a 1 red, 1 green, 2F signal or a 2 red, 2 green IF signal. Nuclei with two Ph chromosomes have either a 1 red, 1 green, 3F signal or a 2 red, 2 green, 2F signal.

4. Ph chromosome-positive intermediate or interphase cells with atypical abnormal I>FISH types will have 5 or fewer signals and at least one fusion signal. Atypical signal types include 2 red and 1 green IF signals, 1 red and 2 green IF signals, or 1 red and 1 green IF signal.

5. If there is a question as to whether a cell should be scored for apricot or whether it has a fusion signal, do not score the cell.

VII. Analysis of specimen preparation

1. Count 100 nuclei as a positive control before analyzing patient samples to ensure that the method works.

2. If possible, analyze 10 or more Ph chromosome-positive interphase divisions before treatment to establish the type of Ph chromosome signal. It is important to know whether to analyze interphase nuclei using typical or atypical scoring criteria.

3. Capture at least two representative interphase phases to characterize these studies.

4. Two observers should each count 250 neighboring interphase nuclei from two or three separate regions of the hybridization site.

5. If the difference between the results of the two observers is greater than 5%, a third observer should count 250 neighboring cells or should consider hybridizing again.

6. If the results of the two observers are acceptable, then these results should be averaged to calculate the percentage of BCR/ABL fusion signaling cells in the sample.

7. If the patient has a Ph chromosome with a typical signal type and the results are within the normal range (less than 1% abnormal nuclei), then more than 2750 nuclei out of 6000 nuclei should be analyzed by each observer to look for the smallest residual foci.

8. Capture an image of at least one area on the slide to depict several nuclei representing the final result.

VIII. Sugamo Explanation

1. Experienced observers should rarely, if ever, observe false-positive signaling types of nuclei in normal samples. For these observers, the normal cut value for 500 nuclei analyzed is less than 1%, and for 6000 nuclei, the cut value is less than 0.079%.

2. Experienced observers should detect more than 90% of abnormal interphase cells in most untreated CML patients.

3. The frequency of false-positive nuclei varies in patients with atypical signal types. 1 red 2 green IF patients have a normal cut value of 1.2%, 2 red 1 green IF patients have a normal cut value of 1.8%, and 1 red 1 green IF patients have a normal cut value of 23.0%.

IX. Quality control

1. Any new reagent should be tested on a control sample before it is used in the clinic.

2. For each batch of patient samples a Ph chromosome positive control slide should be brought.

3. Each sample should be analyzed independently by two recorders whose results should differ from each other within 5%.

4. Tables summarizing the results of each recorder against the samples should be kept and routinely evaluated for trends in technical problems.

5. All failed test results should be investigated and described to establish a problem library.

6. A monthly results report should be generated to evaluate the testing program.

7. Find more information on quality control.

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