| Summary: | Reverse transcriptases (RT) are essential tools in <i>BCR::ABL1</i> fusion transcript monitoring in chronic myeloid leukemia (CML). The RT type and cDNA priming method may impair the stoichiometry of cDNA synthesis, thereby potentially introducing a bias in <i>BCR::ABL1</i> qRT-PCR data. Using the Acrometrix™ <i>BCR::ABL1</i> reference panel and 37 clinical specimens, we have comparatively investigated the performance of the RTs MLV and SuperScript IV with random hexamer vs. target-specific priming. Quantitative RT-PCR results identified the priming type and RT type as major factors for diagnostic data variation, mainly due to the different efficacies of processing <i>BCR::ABL1</i> low-copy-numbers (<50) compared to <i>GUSB</i> or <i>ABL1</i> high-copy targets. The impairment of SuperScript IV in processing low- and high-copy-number RNA targets equally was not reflected by the diagnostically relevant Log (<i>BCR::ABL1/GUSB</i>%) values. Therefore, the correct representation of housekeeping and <i>BCR::ABL1</i> target genes should have priority when aiming at as high a number of housekeeping gene copies as possible. Our data suggest that for improving <i>BCR::ABL1</i> assay sensitivity, increased RNA/cDNA amounts and the use of distinct RT/priming combinations are advantageous. However, for inter-laboratory harmonization, the proper conversion factor according to the CML international standard (IS) has to be reevaluated each time the grade of RT is changed.
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