BCR/ABL, mRNA Detection, Reverse Transcription-PCR (RT-PCR), Qualitative, Diagnostic Assay
Method Description Describes how the test is performed and provides a method-specific reference
RNA is extracted from the patientâ€™s blood or bone marrow obtained at the time of diagnosis and is converted into complementary DNA (cDNA) for ease of analysis. The cDNA is then subjected to PCR using 2 separate reactions: the first contains primers designed to amplify all fusions containing the ABL a2 exon. The second contains primers designed to amplify all fusions containing the ABL a3 exon. The reverse primer in each tube contains a fluorescent label such that all amplified products will be fluorescently labeled. If a bcr/abl fusion product is present in the original sample, this product will be amplified in one or both of these PCR reactions.
The PCR products are then added to a mixture of beads (Luminex Corp.), which is made as follows: for each possible bcr/abl fusion product, a uniquely colored bead is selected and multiple copies of a capture oligonucleotide are bound to its surface. The capture oligonucleotide contains sequence from the BCR portion of the fusion (from the exon closest to the fusion point) such that only a fusion product containing that exon will bind. All of the beads, each coated with their targetâ€™s capture probe, are mixed together and an aliquot is mixed with each of the 2 PCR reactions under annealing conditions. If an amplified bcr/abl target is in the PCR reaction mix, it will bind to its corresponding capture oligonucleotides on its uniquely colored bead. Following annealing, the beads are washed to remove all unbound products and analyzed by the Luminex instrument.
The Luminex instrument uses the principle of flow cytometry and analyzes 1 bead at a time using 2 separate lasers. The first laser identifies the color of the bead, and the second measures the amount of fluorescence on the bead. Any bead coated with captured, fluorescently labeled PCR product will register as high fluorescence. The machine is set to count a specific number of each bead color and a bar graph is generated showing the average fluorescence per bead in each uniquely colored bead set.(Unpublished Mayo method)
Day(s) and Time(s) Test Performed Outlines the days and times the test is performed. This field reflects the day and time the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time required before the test is performed. Some tests are listed as continuously performed, which means assays are performed several times during the day.
Monday through Friday; 2 p.m.