Biomedical Engineering Reference
In-Depth Information
procedures such as amniocentesis and chorionic villus sampling (CVS), from
which the full fetal karyotype is usually determined using cultured cells. The
2-wk period needed for cultivation and subsequent analysis has proven to be
associated with considerable parental anxiety and medical problems in
those situations requiring therapeutic intervention.
In order to address these needs, more rapid methods for the prenatal diagno-
sis of fetal chromosomal aneuploidies have recently been developed and imple-
mented
(
1
)
. The first of these to be commercially introduced was multi-color
fluorescence
in situ
hybridization (FISH) for uncultured cells. Although this
method is very reliable and has proven in large-scale studies to be very accu-
rate, it is a time- and labor-intensive procedure. Furthermore, as this method
requires intact cells, it can only be used on fresh or specially stored samples
(
2-4
)
. The next method that has seen widespread clinical application, particu-
larly in the United Kingdom, is quantitative fluorescent polymerase chain
reaction (PCR) analysis of short tandem repeats (STRs)
(
5-8
)
. This method
has also proven itself to be rapid and reliable, once the initial problems with
polymerase stuttering and amplification failure of the highly repetitive loci had
been overcome.
An important point to bear in mind regarding both of these rapid diagnostic
tests is that they currently only permit a result regarding the most common
fetal aneuploidies (chromosomes X, Y, 13, 18, and 21). Hence, it is still neces-
sary to resort to the normal 2-wk cell culture-based analysis in order to obtain
a full karyotype. Maternal blood contamination of the amniotic fluid or chori-
onic villus sample, although infrequent (affecting less than 2% of samples col-
lected), can interfere with either method of analysis, as the results cannot be
distinguished from cases of fetal mosaicism.
The recent development of real-time PCR has rapidly emerged as a power-
ful tool for the accurate and precise determination of template copy numbers,
and has found widespread applicability in the analysis of gene expression and
cell-free DNA in body fluids as well as the measurement of gene duplications
or deletions in cancer research
(
9-13
)
. The precision of current assays and
technology, however, was not thought to permit analyses of less than twofold
differences of target template concentrations. As our primary interest is in the
development of new methods for prenatal diagnosis, we were interested in
whether this new technology could be used for the determination of fetal triso-
mies. This would, however, entail the resolution of less than twofold incre-
ments in target gene copy number, as in these instances there is only a 50%
increase present in the amount of a particular chromosome.
Our reason for choosing a real-time “TaqMan” PCR approach is that we
have considerable experience with the methodology
(
14-17
)
, especially in the
