J. Meijerink et al., A novel method to compensate for different amplification efficiencies between patient DNA samples in quantitative real-time PCR, J MOL DIAGN, 3(2), 2001, pp. 55-61
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.