Myeloid Leukemia

Real-Time RT-PCR Strategies 59
Ct CG,sample – Ct CG,median; Ct CG,sample is the Ct of the CG in the sample, and
Ct CG,median is the median Ct of the CG in the laboratory. A low ABL expression
level could be due to RNA degradation or to faulty amplification due to the
presence of PCR inhibitors. RNA degradation is not highlighted by the Agilent
2100 Bioanalyzer in the majority of such samples (47). Therefore, the most
likely explanation for low ABL expression is the presence of inhibitors and not
RNA degradation per se. These inhibitors can be removed by dilution of cDNA,
purification of cDNA on columns, or by the addition of an amplification facilitator
such as bovine serum albumin (BSA) in the PCR buffer. It seems that the
addition of 0.04% BSA to TaqMan-based RQ-PCR analysis is better than the
other techniques for achieving satisfactory amplification of transcripts in poor
quality samples (47).
The FG levels in diagnostic samples are also important for calculating the
sensitivity. Indeed, the expression level of an FG in a patient’s diagnostic
sample can vary considerably (up to 100-fold). Furthermore, FG transcripts
have also been detected in the peripheral blood of healthy subjects (30,48). A
simple formula allows calculation of the sensitivity:
dynamic range (log value) = [Ct(FG LIMIT) – Ct(FG DIA)]/slope
where Ct(FG LIMIT) is the Ct limit for detection (the y-intercept if a plasmid
standard curve is used), and Ct(FG DIA) is the Ct of the FG at diagnosis. If the
slope is –3.3 and Ct(FG DIA) is 21, quantitative data are reliable to a dilution of
1 in 10,000. So, to determine the level of MRD in patient samples, the FG
value at diagnosis is set to one, and the fraction of leukemic cells is calculated
from the following equation:
10 x = (∆Ct MRD – ∆Ct DIA)/slope
where x is the fraction of leukemic cells, ∆Ct MRD is the normalized threshold
cycle [Ct(FG MRD) – Ct(CG MRD)] at MRD, and ∆Ct DIA is the normalized threshold
cycle at diagnosis.
The EAC protocol proposes an assessment of the sensitivity of RQ-PCR
experiments. The sensitivity is calculated according to the normalized copy
number (NCN = FG copy number/CG copy number) of the FG at diagnosis
(FG/CG) DIA, and from the level of CG expression (CG MRD) in the follow-up
sample being tested. Accordingly, the sensitivity of RQ-PCR experiments is
(CG CN,DIA/FG CN,DIA × CG CN,MRD). Ideally, the calculation should be based on
the patient’s NCN at diagnosis, after correction for blast percentage. If these
data are not available, EAC values can be used.
In routine practice, the interpretation of patient sample results is dependent
on the control gene expression level to monitor the quality of the sample. Based
on EAC data, samples with ABL CN > 1000 (i.e., 10 ∆Ct/3.3 < 10) are “good