5.2) RAPID ANEUPLOIDY DETECTION
In developed countries, prenatal diagnosis is routinely offered to all women for detection of risk chromosomal abnormalities. In invasive prenatal diagnosis, karyotyping on amniocytes is considered the gold standard in earlier days but it has lost its gold fame due its tedious, labor intensive and costly nature. Furthermore the parents also have to wait for 2 to 3 weeks for the test and the results revealed with karyotyping to detect chromosomal abnormalities are unclear, of mild clinical relevance, so it can cause patient anxiety and emotional dilemmas concerning the continuation of pregnancy so rapid.
Rapid Aneuploidy Detection (RAD) can detect aneuploidies of chromosome 21, 13, 18, X and Y within 1–4 days. RAD shows high accuracy for the detection of these aneuploidies, which account for more than 80% of the clinically relevant chromosomal abnormalities. Besides, the costs are low and rare abnormalities with unclear or mild consequences are not detected.
The advantages of RAD seem less Strong than its promoters suggest. Fast test results only give a short-term psychological benefit. The cost advantage of RAD is apparent, but must be weighed against consequences like missed abnormalities, which are evaluated differently by professionals and pregnant women. The advantage of RAD is that it generates test results within 1–4 days. This is considerably faster than karyotyping, which takes ≥14 days. This reduced waiting time for RAD is clearly advantageous.
Currently available methods for rapid aneuploidy detection (RAD)include fluorescence in situ hybridization (FISH) and quantitative fluorescence polymerase chain reaction (QF-PCR). Multiplexligation-dependent probe amplification (MLPA) is a newer technology under investigation.
FISH and QF-PCR are known to, and MLPA is proving to, havesimilar sensitivity and specificity to full cytogenetic karyotyping (thecurrent “gold standard”) for the detection of fetal aneuploidy (forchromosomes 13, 18, and 21 and the sex chromosomes).
Advantages of QF-PCR and MLPA over full karyotype include substantially reduced turnaround time and automation and batching of samples resulting in reduced cost per sample. FISH is not amenable to automation and remains more costly than PCR-based techniques.
The main disadvantage of current RAD methods is it is failure to detect chromosome aberrations which are not aneuploidy aberration for the targeted chromosomes.
RAD may be a suitable technique for prenatal diagnosis which can reveal about the fetal aneuploidy. It would not be suitable for those women with other risk factors such as structural fetal abnormalities on ultrasound or a personal or family history of a chromosomal rearrangement (such as a balanced translocation), for these women a full cytogenetic karyotype analysis is advised so that complete cytogenetic analysis can be done.
For non-mosaic standard trisomy, cultured karyotype analysis has been considered a reliable detector of fetal abnormality. However, the sensitivity of karyotyping depends on the number of cells established in a particular culture, and results are usually not available for 3-4 days or more. Furthermore, it is very difficult to identify chromosome microdeletions. In addition to karyotype analysis, fluorescence in situ hybridization (FISH), an easy-to-handle, rapid, and highly sensitive tool for genetic analysis, has been developed in the past two decades. Recently, AneuVysion FISH analysis has become the most common rapid screening method for prenatal or neonatal aneuploidies in a clinical setting. In most laboratories, AneuVysion analysis for prenatal testing costs between $300 and $350. However, FISH is labor-intensive and not easily applicable to a large number of samples in clinical diagnostic settings.
Quantitative fluorescence PCR (QF-PCR) provides the possibility to detect copy number variation of chromosomal sequences in several hours. It also has the advantage of being much cheaper and allowing the simultaneous processing of larger numbers of samples than FISH and karyotyping analysis. However, the presence of multiple primer pairs in a PCR reaction reduces the reliability of the quantification. To solve these technical problems, multiplex ligation-dependent probe amplification (MLPA) has emerged as an alternative to standard PCR-based techniques for detection of the chromosome aneuploidies. It allows for relative quantification of up to 50 different target sequences in one reaction and does not require living cells or cell culture. It is less labor-intensive and less expensive compared to karyotyping and FISH. Therefore, MLPA has been widely applied for molecular diagnosis of genetic diseases such as DMD, Spinocerebellar ataxia type 15 and chromosomal aneuploidies.