Deletions of the long arm of chromosome 5 are one of the most common karyotypic abnormalities reported in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) with myelodysplasia related changes1,2. EGR1 (early growth response 1), a tumor suppressor gene at 5q31.2, has been shown to act through haploinsufficiency to initiate the development of MDS/AML3. Loss of 5q31.2, the region detected by this probe set, which includes the EGR1 gene, have been linked to a more aggressive form of MDS and AML and is often accompanied by additional cytogenetic abnormalities and a poorer prognosis2,4,5. This probe can also detect some deletions that are associated with 5q- syndrome2. However, the probe does not cover the critical deleted region for 5q33 and is not intended for the detection of all deletions associated with 5q- syndrome.
The Del(5q) Deletion FISH Probe Kit is a fluorescence in situ hybridization (FISH) Test used to detect deletions within the long arm of chromosome 5 at location 5q31.2 in fixed bone marrow specimens from patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). 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.
Product availability may vary from country to country and is subject to varying regulatory requirements. Please contact your local representatives for availability.
1. Swerdlow et al., (eds,) WHO Classification of Tumours of Haematopoietic and Lymphoid Tissue, Lyon, France, 4th edition, IARC,2017.
2. Ebert BL., Best Pract Res Clin Haematol. Netherlands; 2010 Dec;23(4):457–61.
3. Boultwood J et al., Blood. 2010;116(26):5803–11.
4. Joslin JM et al., Blood. 2007 Jul 15;110(2):719–26.
5. Fang J et al., Cell Rep. 2014;8(5):1328–38.
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.
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