Myeloid Leukemia

Quantitative PCR for Monitoring CML Patients 87
Table 1
The Inter-Assay Variation of Threshold Cycle (Ct) Values for
the Six b3a2 BCR-ABL Standards Measured Over 2 Yr (n =
134) and Using a Threshold of 0.04
Copy number Mean standard 2 standard Coefficient of
of standards Ct value deviation range variation (%)
15 36.45 35.36–37.54 1.50%
1.5 × 10 2 33.01 32.35–33.67 1.00%
1.5 × 10 3 29.53 28.90–30.16 1.06%
1.5 × 10 4 26.12 25.61–26.63 0.98%
1.5 × 10 5 22.75 22.20–23.30 1.21%
1.5 × 10 6 19.37 18.96–19.78 1.05%
samples in duplicate apart from the lowest standard, as a result of the wider variability
at high threshold cycle (Ct) levels. The inter-assay reproducibility of the
standard values is usually within 1.3 Ct, except for the lowest standard (see Table 1).
The 2 standard deviation range provides an estimate of measurement reliability
of the Ct values at approx the 95% level of confidence. The patient and control
results are calculated from the standard curve and printed on the experiment
report. Before accepting the results, the fluorescent curve of each control and
patient sample must be viewed to ensure that the curve is linear and therefore
acceptable. For some patients with BCR-ABL values that are not detectable, the
background fluorescence may “creep” above the threshold. If this occurs, the
quantitative value will be calculated from the fractional Ct value at the point it
crosses the threshold. Therefore, a very high quantitative value will be assigned
instead of a negative value. These samples must be noted and the correct value
assigned on the experiment report. The HeLa negative control does on occasion
produce a positive value that is less than 10 copies, in which case it is assigned as
not detected. Patient values that are below this level are also assigned as not
detected. If the negative control is greater than 10 copies, contamination may have
occurred, and the samples are repeated. If the contamination represents only a very
small number of copies, it will not influence the result of patients with values that
are greater than 2 logs higher, and these samples need not be repeated. We have
identified what appears to be a software error for the no-template control result. If
these samples are assigned as “NTC” during sample setup, the result will always be
printed as zero on the experiment report even for positive values (see arrows in Fig.
3). Therefore, the no-template control fluorescence data must be viewed to ensure
that it is actually negative. The BCR control transcript value provides an indication
of the quality of the RNA. Values range from 30,000 to greater than 300,000 transcripts
per 2.5 µL of cDNA for acceptable quality. Below 30,000 transcripts, the
RNA is considered degraded and a repeat sample is requested. The RQ-PCR procedure,
including processing the RNA extraction batch, the reverse transcription PCR,