Use of Real-Time Polymerase Chain Reaction for the Detection of Fetal Aneuploidies - Clinical Applications of PCR - page 91

Table 1
The Calibrator Values for the Three Karyotypes
and the Four Thresholds Used a

∆ C T calibrator values

Threshold 0.2 0.3 0.45 0.675
Trisomy 18 -0.52 -0.55 -0.68 -0.73
Normal 0.01 -0.03 -0.09 -0.11
Trisomy 21 0.48 0.45 0.44 0.42
a The average ∆C T -values of the control sample measurements
for each karyotype are the respective calibrator values.

sible as a result of the poor quality or inadequate quantity of the DNA sample
analyzed.
In the interim period, Applied Biosystems has introduced the smaller, less
costly and user-friendlier SDS 7000 to the market. This instrument obviates
the need for a complex laser-based system, instead employing a halogen lamp
for the excitation of fluorescent probes. After careful appraisal of this instru-
ment, which we had determined to be as robust, reliable, and accurate as the
more sophisticated and larger SDS 7700, we then adapted our assay in such a
manner that it would permit the simultaneous analysis of trisomies 18 and 21.
This was achieved by replacing the chromosome 12 ( glyceraldehyde-3-phos-
phate dehydrogenase [ GAPDH ] gene) control amplicon of the original trisomy
21 assay with a sequence on chromosome 18. In order to increase the specific-
ity of our new assay, we also examined the influence of various other param-
eters, such as PCR reaction mix composition and primer sequences and purity
as well as different probe chemistries. These studies were undertaken to ensure
that the amplification efficiencies of the two reactions (chromosome 18 and
21) were equal.
As described previously, reference calibration values for the two chromo-
somes being interrogated were determined by examining DNA samples with
either normal, trisomy 18, or trisomy 21 karyotype ( Table 1 ). Once we had
established these reference calibration values, we performed a large-scale study
of almost 100 clinical amniotic fluid samples, in which we examined the ploidy
for these two chromosomes in a blinded manner (Zimmermann et al., manu-
script in preparation). In more than 86% of the cases, the aneuploidy status for
these two chromosomes was established correctly. In those instances where we
were not successful, the analysis was either hindered by inadequate concentra-
tions of template DNA or by the presence of inhibitors in the original DNA
preparation ( see Note 7 ).
These results clearly demonstrate the diagnostic potential of real-time PCR,
whether for prenatal diagnosis or for other analyses involving gene duplica-