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From the Department of Pediatrics,
*
Division of Oncology/Hematology, Sophia Children’s Hospital, Erasmus University Rotterdam, Rotterdam, The Netherlands; the Department
and Central Laboratory
of Hematology, University Hospital Nijmegen, Nijmegen, The Netherlands; and Applied Biosystems, Inc.,
Foster City, California
Quantification of residual disease by real-time polymerase chain
reaction (PCR) will become a pivotal tool in the development of
patient-directed therapy. In recent years, various protocols to
quantify minimal residual disease in leukemia or lymphoma patients have
been developed. These assays assume that PCR efficiencies are equal for
all samples. Determining t(14;18) and albumin reaction efficiencies for
sixteen follicular lymphoma patient samples revealed higher
efficiencies for blood samples than for lymph node samples in general.
However, within one sample both reactions had equivalent
efficiencies. Differences in amplification efficiencies between patient
samples (low efficiencies) and the calibrator in quantitative analyses
result in the underestimation of residual disease in patient samples
whereby the weakest positive patient samples are at highest error.
Based on these findings for patient samples, the efficiency
compensation control was developed. This control includes two reference
reactions in a multiplex setting, specific for the ß-actin
and albumin housekeeping genes that are present in a constant ratio
within DNA templates. The difference in threshold cycle values for both
reference reactions, ie, the 
Ct2
value, is dependent on the amplification efficiency,
and is used to compensate for efficiency differences between patient
samples and the calibrator. The ß-actin reference reaction is also
used to normalize for DNA input. Furthermore, the efficiency
compensation control facilitates identification of patient samples that
are so contaminated with PCR inhibitory compounds that different
amplification 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 retrospective studies, but
even more so, in future molecular diagnostic
analyses.
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