Objective:We present the results of analytical verification and our experience with clinical use of real-timePCR for relative quantification of BCR-ABL RNA transcripts. Setting: Institute of Clinical Biochemistry and Diagnostics, Charles University Hospital, Hradec Králové, Czech Republic; Department of Clinical Hematology, Charles University Hospital, Hradec Králové, Czech Republic. Material and Methods: A total of 90 peripheral blood and bone marrow specimens were analysed. For real-time PCR the LightCycler t(9; 22) Quantification Kit, based on hybridization probe chemistry, was used. The number of BCR-ABL transcripts in clinical samples was normalized in ratio to the amount of G6PDH transcripts and the results were expressed in percentages. Results: The group of “strongly positive“ samples (N = 44), determined semi-quantitatively by single-roundPCR (SQ-PCR), varied in BCR-ABL quantity from 0.08% up to 50.55%, with a median of 3.40%. The SQ-PCR cohort of “nested-round positive“ samples (N = 27) fluctuated between 0% and 0.12% with a median of 0.01%. No BCR-ABL transcripts were found in “nested-round negative“ samples (N = 19). The lowest, but surely detectable, fluorescent signal was recorded in the dilutionsample withBCR-ABLquantity of 0.001%.Thespecificity was 100%.Theoverall variability of the used method in clinical samples was 25%. Conclusions: Despite single-step amplification, real-time PCR is a sensitive laboratory method that, compared to SQ-PCR, has provided concordant and reproducible results in clinical samples where BCR-ABL levels were higher than 0.001%.

        Key words: BCR-ABL, quantification, real-time PCR, leukemia, RNA.