MLL (KMT2A) Breakapart
- Applications
- hematology
- Area of Interest
- AML, MDS
- Catalogue Numbers
- USA-LPH 013 (10 tests)
Probe Specification
- MLL, 11q23.3, Texas Red
- MLL, 11q23.3, FITC Green
The MLL (KMT2A) Breakapart FISH Probe Kit consists of an 87kb probe, labeled in Texas Red, covering a region telemetric to the MLL (KMT2A) gene including the marker SHGC-111513 and a FITC green probe covering a 170kb region centromeric to the MLL (KMT2A) gene spanning the CD3G and UBE4A genes.
Probe Information
The KMT2A (lysine methyltransferase 2A) gene at 11q23.3 encodes for a histone methyltransferase, which functions as an epigenetic regulator of transcription1. KMT2A rearrangements are reported frequently in patients with AML and have also been reported in patients with therapy related MDS, albeit at a lower frequency2,3,4,5,6. Historically, KMT2A rearrangements in acute leukemia were associated with a poorer outcome, but recent studies have shown that the prognosis is highly dependent on the fusion partner and may differ between children and adults7. Because this is a breakapart probe, it cannot be used to determine the fusion partner.
Recommended Protocol
Intended Use
The MLL (KMT2A) Breakapart FISH Probe Kit is a fluorescence in situ hybridization (FISH) Test used to detect rearrangement of the MLL (KMT2A) region on chromosome 11 at location 11q23.3 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.
For sale in the US only. This product has not been licensed in accordance with Canadian law.
References
1. Tomizawa D., Pediatr Int. 2015;4:811–9.
2. Wright RL et al., Crit Rev Oncol Hematol. 2014;91(3):283–91.
3. van der Burg M et al., Leuk Off J Leuk Soc Am Leuk Res Fund, UK. 2004;18(5):895–908.
4. Grossmann V et al., Leukemia. England; 2013. p. 1933–6.
5. Super HJ et al., Blood. United States; 1993 Dec;82(12):3705–11.
6. Schanz J et al., J Clin Oncol. 2012;30(8):820–9.
7. Tamai H et al., J Clin Exp Hematop. 2010;50(2):91–8.
Microscope Images
Recommended Protocol and Sample Preparation
1Sample and Slide 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.
2Pre-denaturation
- 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.
3Denaturation
- Denature the sample and probe simultaneously by heating the slide on a hoplate at 75°C (+/- 1°C) for 2 minutes.
4Hybridization
- Place the slide in a humid, lightproof container at 37°C (+/- 1°C) overnight.
5Post-Hybridization Washes
- 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.
6Analyze
- 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