Several potential pitfalls must be avoided to prevent false-positive and false-negative results with translocation RT-PCR assays. First, RNA degradation must be prevented by careful specimen handling. Second, amplification of a control mRNA from the same cDNA preparation used for amplification of the fusion transcript is used to control for the integrity of the RNA, the quality of the RT step, and the presence of inhibitors.
A major concern has been raised about the clinical utility of RT-PCR MRD translocation testing in ALL
patients because fusion transcripts can be detected in the hematopoietic tissues of healthy individuals. Low levels of BCR/ABL transcripts have been detected in a sizable proportion of healthy individuals.87 Using a two-round (nested) RT-PCR method, the MLL/AF4 fusion transcript has been detected in the fetal liver and BM of normal infant BM samples and in 12% of pediatric ALL with no cytogenetic or genomic evidence of 11q23 alterations.88 The significance of these findings is still elusive.
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