Signature LTx v2.0

Signature^® LTx v2.0 Leukemia Translocation Panel

The Signature^® LTx v2.0 CE IVD Kit (Leukemia Translocation Panel) is a qualitative in vitro diagnostic device for use in a clinical laboratory to identify specific fusion transcripts in total RNA from whole blood or bone marrow to aid in the clinical diagnosis of translocation positive leukemias. The assay is a multiplex reverse transcription PCR (RT-PCR) amplification, followed by multiplex amplicon detection on the Luminex^® 100^™ IS or 200^™ system.

The assay is not intended for stand-alone diagnostic purposes or for minimal residual disease monitoring.

The assay is not intended to detect fusion transcripts resulting from breakpoints other than the ones listed in Table 1.

Figure 1. Overview of the Signature® LTx v2.0 assay principle and workflow.

The Signature^® LTx v2.0 assay combines multiplex RT-PCR with multiplex fluorescent bead-array detection (Figure 1). The assay is designed to simultaneously amplify the fusion transcripts listed in Table 1 and an endogenous control GAPDH in a single test. Positive and negative control samples are provided in the kit to assess the validity of each run. The assay provides comprehensive results delivered via its streamlined workflow with minimal hands-on time. The entire procedure can be completed in less than six hours for up to 24 reactions per kit.

Table 2: Simultaneous Detection of 12 Leukemia-Associated Fusion Transcripts and Endogenous Control GAPDH

Signature^® LTx Negative, Positive and no target (NTC) controls were analyzed together with specimens consisting of 100000 copies of fusion transcripts prepared by in vitro transcription and spiked into a background of 400 ng of translocation-negative cell line RNA (HL-60). Qualitative results are measured by MFI (mean fluorescent intensity). MFI values greater than or equal to 350 MFI signify positive detection for a specific fusion transcript. A sample is considered negative if the MFI value is below 350 MFI and the GAPDH signal is greater than or equal to 1000 MFI. All 12 fusion transcripts and the endogenous control GAPDH were appropriately detected. No cross reactivity between the various targets was observed and MFI values for all non-target probes were reproducibly below the cut-off (350 MFI).

Figure 2: Serial Dilution of Cell Line Total RNA

In this experiment, analytical sensitivity was evaluated by diluting total RNA purified from translocation positive leukemic cell lines into a background of total RNA purified from a translocation negative cell line (HL-60) and keeping the total RNA input constant at 400 ng. Only MFI results for AML1/ETO, TEL/AML1 and the GAPDH probes are shown. As different cell lines express different levels of fusion transcripts, TEL/AML1 and AML1/ETO fusion transcripts were detected when the translocation positive cell line RNA represented as low as 1 or 0.1% of the total RNA input, respectively. This analytical sensitivity is equivalent to the detection of 1 fusion transcript positive cell in a background of at least 100 to 1000 fusion transcript negative cells. The GAPDH endogenous control was simultaneously detected in all specimens except the no template control (NTC).

References:

Gabert J, Beillard E, van der Velden VH, Bi W, Grimwade D, Pallisgaard N, Barbany G, Cazzaniga G, Cayuela JM, Cave H, Pane F, Aerts JL, De Micheli D, Thirion X, Pradel V, Gonzalez M, Viehmann S, Malec M, Saglio G, van Dongen JJ. Standardization and Quality Control Studies of “Real-Time” Quantitative Reverse Transcriptase Polymerase Chain Reaction of Fusion Gene Transcripts for Residual Disease Detection in Leukemia – A Europe Against Cancer Program. Leukemia. 17(12):2318-57. 2003.

Salto-Tellez M, Shelat SG, Benoit B, Rennert H, Carroll M, Leonard DG, Nowell P, Bagg A. Multiplex RT-PCR for the Detection of Leukemia-Associated Translocations: Validation and Application to Routine Molecular Diagnostic Practice. J Mol Diagn. 5(4):231-6. 2003.

Bock O, Reising D, Kreipe H. Multiplex RT-PCR for the Detection of Common BCR-ABL Fusion Transcripts in Paraffin-Embedded Tissues from Patients with Chronic Myeloid Leukemia and Acute Lymphoblastic Leukemia. Diagn Mol Pathol. 12(3):119-23. 2003.

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