AML1/ETO (RUNX1/RUNX1T1) Translocation, Dual Fusion
- ETO, 8q21.3, FITC Green
- AML1, 21q22.1, Texas Red
The AML1/ETO (RUNX1/RUNX1T1) Translocation, Dual Fusion FISH Probe Kit consists of a 156kb probe labeled in Texas Red, centromeric to the AML1 (RUNX1) gene, including the CLIC6 gene; a 169kb probe labelled in Texas red, telomeric to AML1 (RUNX1) gene, extending beyond the marker D21S1921; and two (151kb and 194kb) probes, labeled inn FITC green, on either side of the ETO (RUNX1T1) gene.
AML with a RUNX1-RUNX1T1 fusion resulting from a t(8;21)(q22;q22) translocation is a recognized disease entity according to the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia1. The translocation is commonly observed in patients with AML FAB type M2, most commonly in children and young adults2 and is a good prognostic indicator3,4,5. The t(8;21) breakpoint mainly occurs in the intron between exons 5 and 6, just before the transactivation domain. The fusion protein created contains the DNA-binding domain of RUNX1 fused to the transcription factor RUNX1T12. In addition to the reciprocal t(8;21) translocation creating the RUNX1-RUNX1T1 fusion, variant translocations have also been reported. These variant rearrangements may be cryptic and easily overlooked by G-banding; however, FISH can indicate the presence of such rearrangements2.
The AML1/ETO (RUNX1/RUNX1T1) Translocation, Dual Fusion FISH Probe Kit is a fluorescence in situ hybridization (FISH) Test used to detect rearrangement involving the AML1 (RUNX1) region on chromosome 21 at location 21q22.1 and the ETO (RUNX1T1) region on chromosome 8 at location 8q21.3 in fixed bone marrow specimens from patients with acute myeloid leukemia (AML). The test is indicated for characterization of patient specimens consistent with World Health Organization (WHO) guidelines for Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised 4th Edition) and in conjunction with other clinicopathological criteria. The assay results are intended to be interpreted by a qualified pathologist or cytogeneticist. The test is not intended for use as a stand-alone diagnostic, disease screening, or as a companion diagnostic.
Limitations of the Procedure
For In Vitro Diagnostic Use. Rx only.
Reporting and interpretation of FISH results should be consistent with professional standards of practice and should take into consideration other clinical and diagnostic information. This kit is intended as an adjunct to other diagnostic laboratory tests and therapeutic action should not be initiated on the basis of the FISH result alone. Failure to adhere to the protocol may affect the performance and lead to false results.
Each lab is responsible for establishing their own cut-off values. Each laboratory should test sufficiently large number of samples to establish normal population distribution of the signal levels and to assign a cut-off value. The product is for professional use only and is intended to be interpreted by a qualified Pathologist or Cytogeneticist.
The device has not been specifically validated in patients with <20% blast count.
For sale in the US only. This product has not been licensed in accordance with Canadian law.
1.Swerdlow et al., (eds,) WHO Classification of Tumours of Haematopoietic and Lymphoid Tissue, Lyon, France, 4th edition, IARC,2017.
2. Reikvam HH et al., J Biomed Biotechnol. 2011;2011:1–23.
3. Grimwade D et al., Blood. 2001 Sep 1;98(5):1312–20.
4. Harrison CJ et al., J Clin Oncol. 2010;28(16):2674–81.
5. Grimwade D et al., Blood. 2010;116(3):354–65.
Recommended Protocol and Sample Preparation
- The FISH probes for AML/MDS are designed for use on bone marrow cells fixed in Carnoy’s solution (3:1 methanol/ acetic acid) that are prepared according to the laboratory or institution guidelines.
- Spot the cell sample onto a glass microscope slide. Allow to dry.
- Immerse the slide in 2x Saline Sodium Citrate (SSC) for 2 minutes at room temperature (RT) without agitation.
- Dehydrate in an ethanol series (70%, 85% and 100%), each for 2 minutes at RT.
- Allow to dry.
- Remove the probe from the freezer and allow it to warm to RT. Briefly centrifuge tubes before use.
- Ensure that the probe solution is sufficiently mixed with a pipette or a vortex mixer.
- Remove 10μl of probe per test, and transfer it to a microcentrifuge tube. Quickly return the remaining probe to -20°C.
- Place the probe and the sample slide to prewarm on a 37°C (+/- 1°C) hotplate for 5 minutes.
- Spot 10μl of probe mixture onto the cell sample and carefully apply a 24x24 mm coverslip. Seal with rubber solution glue and allow the glue to dry completely.
- Denature the sample and probe simultaneously by heating the slide on a hoplate at 75°C (+/- 1°C) for 2 minutes.
- Place the slide in a humid, lightproof container at 37°C (+/- 1°C) overnight.
- Remove the DAPI from the freezer and allow it to warm to RT.
- Remove the coverslip and all traces of glue carefully.
- Immerse the slide in 0.4x Saline Sodium Citrate (SSC) (pH 7.0) at 72°C (+/- 1°C) for 2 minutes without agitation.
- Drain the slide and immerse it in 2xSSC + 0.05% Tween-20 at RT (pH 7.0) for 30 seconds without agitation.
- Drain the slide and apply 10μl of DAPI antifade onto each sample.
- Cover with a 24x24mm coverslip, remove any bubbles.
- Edge the slide with clear nail varnish to seal.
- Allow the color to develop in the dark for 10 minutes.
- View with a fluorescence microscope.
- For optimal visualization of the probes, a 100-Watt mercury lamp (or equivalent) is recommended with plan apochromat objectives 63x or 100x.
- Filters designed specifically for detection of DAPI, FITC, Texas Red®, and Aqua or DEAC fluorophores individually or in combination (e.g. dual or triple filters) are optimal for best results.
- The final hybridized slides are analyzable for up to 1 month when stored in darkness and at 2-8°C.
For additional support and advice about the FISH technique please visit our support page