A novel method to compensate for different amplification efficiencies between patient DNA samples in quantitative real-time PCR

Quantification of residual disease by real-time polymerase chain reaction ( PCR) will become a pivotal tool in the development of patient-directed ther apy, In recent years, various protocols to quantify minimal residual. disea se in leukemia or lymphoma patients have been developed. These assays assum e that PCR efficiencies are equal for all samples, Determining t(14;18) and albumin reaction efficiencies for sixteen follicular lymphoma patient samp les revealed higher efficiencies for blood samples than for lymph node samp les in general, However, within one sample both reactions had equivalent ef ficiencies. Differences in amplification efficiencies between patient sampl es (low efficiencies) and the calibrator in quantitative analyses result in the underestimation of residual disease in patient samples whereby the wea kest positive patient samples are at highest error. Based on these findings for patient samples, the efficiency compensation control was developed. Th is control includes two reference reactions in a multiplex setting, specifi c for the p-actin and albumin housekeeping genes that are present in a cons tant ratio within DNA templates. The difference in threshold cycle values f or both reference reactions, ie, the Delta Ct(2) value, is dependent on the amplification efficiency, and is used to compensate for efficiency differe nces between patient samples and the calibrator. The beta -actin reference reaction is also used to normalize for DNA input. Furthermore, the efficien cy compensation control facilitates identification of patient samples that are so contaminated with PCR inhibitory compounds that different amplificat ion reactions are affected to a different extent. Accurate quantitation of residual disease in these samples is therefore impossible with the current quantitative real-time PCR protocols. Identification and exclusion of these inadequate samples will be of utmost importance in quantitative retrospect ive studies, but even more so, in future molecular diagnostic analyses.