Response: Reliability of PCR for BCR-ABL transcripts

We agree with the statements made by Hertzberg and McDonald regarding the reliability of the measurements at different levels that are acceptable over the dynamic range to account for the wider variability at very low levels of BCR-ABL transcripts. A stratification of assay variability has previously been demonstrated over the dynamic range; it was 4.5-fold at a value representing 0.01% and more than 2-fold for values above 0.1%.¹  Our paper emphasized that a more than 2-fold rise occurring between values equating to 0.01% and 0.1% may fall within the coefficient of variance (CV) of the assay and would not then have biologic significance. Our more recent paper²  also suggested that to maximize reproducibility and to enhance detection of low BCR-ABL transcript levels, the ideal would be to divide the sample into 2 aliquots and assay both samples separately. Each analysis would include both the reverse transcriptase (RT) and the quantitative polymerase chain reaction (PCR) stages. Therefore, our recommended procedure already does take into account the possible impact of interassay variability that Hertzberg and McDonald address.

The suggestion from Hertzberg and McDonald that including the previous RNA sample for each patient will mitigate the effect of interassay variability is of interest. One must remember, however, that thawing and retesting RNA open the door to degradation during storage and during thawing. Degradation may introduce additional variability of the result since BCR-ABL levels will be lowered in this situation. The authors need to demonstrate that their suggested method is more reliable for identifying a true rise rather than merely to use appropriate criteria based on the CV of the assay, which reflects the varying measurement reliability across the dynamic range. Thus, the Hertzberg and McDonald suggestion may prove valuable, but before it is accepted as a major advance it would be useful to confirm its superior ability to recognize a true rise in BCR-ABL.