Results of a prenatal aneuploidy screening in uncultured amniocytes using FISH are available within a few hours. Thus, this so-called prenatal quick test is nowadays routinely and successfully applied to reduce psychological distress for pregnant women. The application of molecular cytogenetics for the prenatal screening of chromosomal aneuploidies overall is very reliable and has, compared with other (e.g., PCR-based) approaches, the advantage that analyses are performed on the single-cell level. Thus, cellular mosaics can also be picked up. Even though there are tendencies to apply the prenatal screening of chromosomal aneuploidies exclusively, we strongly support the recommendation of the German Society of Human Genetics, that this kind of test should only be used as a preamble to full chromosome analysis by microscopy to avoid pitfalls.
Rapid, steadfast and early diagnostics is the main concern in prenatal medicine. The evaluation of sonographic markers such as nuchal translucency and biochemical parameters in maternal serum free β-human chorionic gonadotropin (β-HCG) and pregnancy-associated plasma protein (PAP)-A, enables noninvasive first trimester risk screening Citation[1]. In addition to the well-established chorionic villi, short-term culture in invasive prenatal diagnosis interphase FISH on uncultured amnion cells has become a reliable tool for the rapid detection of fetal aneuploidies. According to Stummerl et al., FISH is a quick, inexpensive, accurate, sensitive and relatively specific method for aneuploidy detection in samples of uncultured chorionic villus cells and amniotic fluid cells Citation[1]. This molecular cytogenetic approach is nowadays applied for the detection of the autosomal trisomies 13, 18 and 21 as well as for X and Y abnormalities, which are the major aneuploidies diagnosed prenatally Citation[1]. Thus, 80–95% of all chromosomal disorders expected in the second trimester of pregnancy can be discovered within 24 h or less Citation[2–3].
At present, it takes around 12.5 days to obtain a full fetal karyotype from amniocytic fluid Citation[4,5]. Even though this is relatively short compared with the 3–4 weeks necessary in the 1980s, it is recognized that such waiting times cause psychological distress for pregnant women Citation[6]. This was the major reason for the introduction of molecular cytogenetics methods for prenatal diagnosis of the most common chromosome disorders. As in aforementioned approaches, no time consuming cell culture is required and results can routinely be obtained within 24 h or even less. Besides FISH, quantitative fluorescence (QF)–PCR is a method of choice in order to save time Citation[6].
Available probe sets
The only US FDA-approved FISH test for rapid aneuploidy screening in uncultivated amniocytic cells is the Aneu Vysion kit (commercially available from ABBOTT/Vysis) consisting of three α-satellite DNA probes for chromosomes X, Y and 18 (cep X, cep Y and cep 18) and two locus specific probes for 13q14 (LSI 13) and 21q22.13∼22.2 (LSI 21). The three centromeric probes and the two locus specific probes are applied in two different hybridizations to the samples. Using this probe set it is possible to detect trisomy 13, 18, 21, triploidy, monosomy X and other gonosomal aneuploidies. Unbalanced structural rearrangements, balanced structural rearrangements numerical aberrations beside 13, 18, 21, X or Y, microdeletions/microduplications, uniparental disomy and mutations remain detectable only by molecular genetic methods.
Another probe set called PrenatScreen is offered from Kreatech Diagnostics with a similar probe combination as well as a so-called PloidyScreen that contains probes for chromosome 21, X and Y.
Evaluation of the rapid aneuploidy screening
For rapid aneuploidy screening it is necessary to prepare the uncultivated amniocytes as recommended in the Aneu Vysion kit protocol. A total of 50 interphase nuclei per case and probe combination are evaluated under the fluorescence microscope. This is a semistatistic evaluation. Cut-off rates for normal and abnormal results as well as interpretation suggestions for intermediate results were provided Citation[5]. Here, hints on gender-specific interpretation are also given, which take into account the possibility of maternal cell contamination Citation[5]. However, each laboratory working with rapid aneuploidy screening is encouraged to set up its own cut-off rates. Furthermore, we strongly recommend that the number of analyzed nuclei is increased if the result for aneuploid cells is around the cut-off value.
Pitfalls
According to Stumm et al. and our own experience with approximately 3000 tests in the last 10 years, only approximately 2% of chromosomal aberrations are not detectable by the Aneu Vysion-test system Citation[1]. However, always approximately 5% are consistently ‘problematic cases’ due to inadequate cell numbers, maternal cell contamination or hybridization failure. In most of these cases an adequate FISH result is still obtainable by repeating FISH on the same slide or by analyzing floating amnion cells from the supernatant in cell culture flasks. Thus, in the overwhelming majority of the studied cases, the results obtained by rapid aneuploidy test and banding cytogenetics are in concordance. However, there are a handful of cases that led to false-positive or false-negative results due to centromeric polymorphisms, the presence of a small supernumerary marker chromosome or a dicentric marker chromosome not detectable in the interphase Citation[1,5,7–9]. In summary, the rapid aneuploidy test has a reliability of more than 99.8% Citation[1,5].
Most, if not all, observed pitfalls and misdiagnoses of the prenatal aneuploidy screening by FISH could easily be omitted by the exclusive use of locus-specific probes Citation[7]. The advantage of using probes such as those that are already being applied (LSI 13 and LSI 21), was proven, for example, by the fact that the LSI 21 probe is suited to distinguish between free and translocation trisomy 21 in the interphase Citation[10].
Conclusion
In summary, the rapid prenatal aneuploidy test is – if applied with the necessary sensibility and by careful explanation of its possibilities and limitations – a powerful tool for the clinician in the care of pregnant women.
Financial & competing interests disclosure
Supported in parts by the German Academic Exchange Service (DAAD; D07/00070). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.
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