Estudio de efectividad diagnóstica de la reacción en cadena de la polimerasa cuantitativa fluorescente en fetos con cribado prenatal alterado
The prenatal diagnosis of chromosomal abnormalities had a great evolution over time. It began with the first chromosomal cell culture in amniotic fluid in 1966, and was initially used to identify numerical chromosomal abnormalities, particularly Trisomy 21. With the advent of cytogenetic banding tec...
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| Formato: | Tesis de maestría acceptedVersion |
| Lenguaje: | Español |
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Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica
2023
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| Acceso en línea: | http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_8030 https://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_8030.dir/8030.PDF |
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| Sumario: | The prenatal diagnosis of chromosomal abnormalities had a great evolution over time.
It began with the first chromosomal cell culture in amniotic fluid in 1966, and was initially used to identify numerical chromosomal abnormalities, particularly Trisomy 21. With the advent of cytogenetic banding techniques, fundamental chromosomal abnormalities began to be detected.
In this way, the practice of the karyotype in the amniotic cells between 15 and 17 weeks of gestation, became the primary cytogenetic technique in prenatal diagnosis for two decades, but the cytogenetic diagnosis was confined to the second trimester of gestation. In the early 1980s, prenatal diagnosis during the first trimester was introduced, incorporating chorionic biopsy starting at 12 weeks of gestation, and this led to early identification of fetal chromosomal abnormalities in the first trimester, with earlier diagnosis.
First, the invasive test was indicated in all those with advanced maternal age, due to the connection between aneuploidies a consecuence of a meiotic nondisjunction event and maternal age, but this strategy only identified 30% of trisomies, with a very high number of invasive tests with normal results and a large subdiagnosis of aneuploidies.
With the improvement of screening techniques for patients at risk, ultrasound screening strategies with highly sensitive sonographic markers were determined, which increased the aneuploidy detection rate by up to 90%. Adding biochemical markers in maternal blood (incorporation of free chorionic gonadotropin subunit dosage and protein A of pregnancy), increased the aneuploidy detection rate to 95%.
In recent years, advanced non-invasive screening for the detection of aneuploidies in cell free fetal DNA in maternal circulation has been incorporated, raising the detection rate to 99%, with a false positive rate of 0.01%.
Thus, by implementing these screening methods, the number of invasive procedures was significantly reduced and it was possible to identify patients at high risk of aneuploidy.
Once the prenatal material has been obtained through an invasive procedure, the samples need to be cultured for several days to obtain a cell division that allows stopping the process in metaphase, to then make sample of the chromosomes with different cytogenetic techniques that allow their analysis under an optical microscope. This prenatal karyotyping process takes approximately 15-21 days for amniotic fluid and 7-10 days for chorionic biopsy culture (short or direct culture and long culture). This delay in the result generates a lot of anguish and uncertainty in the parents, and in the health team, when waiting for a result to define a prognosis, behavior for fetal treatment, or define the need for other fetal prenatal studies.
For this reason, to shorten the waiting time for the result, the rapid diagnosis technique for aneuploidies was incorporated, called fluorescent quantitative polymerase chain reaction or QF-PCR. This molecular technique evaluates STRs that allows evaluating aneuploidies of chromosomes 21, 18, 13, X and Y within 24 hours of extracting the prenatal sample.
If the QF-PCR is normal, the karyotype allows evaluation of the other chromosomes, and if it is altered, the karyotype allows confirming whether it is a free trisomy or the product of a structural rearrangement.
When the pregnancy has a high risk of aneuploidies, or fetal malformations, and the QF-PCR and karyotype studies were normal, there is a comparative genomic hybridization microarray study that allows the investigation of DNA microdeletions and duplications with a resolution 100 times higher than that of karyotype.
The main objective was to establish the most effective genetic diagnostic strategies for fetuses with altered screening and/or with congenital malformations, which are diagnosed prenatally, combining screening methods, cytogenetic and molecular diagnostic techniques.
The secondary objective was to evaluate and compare the diagnostic performance of the QF-PCR in comparison with the conventional cytogenetic technique to identify advantages, limitations, challenges of counseling after its implementation.
The present study included 596 prenatal samples that underwent prenatal karyotype and QF-PCR. The diagnostic performance for the detection of aneuploidies 21, 18, 13, X and Y was comparable, without significant differences, with the advantage of rapid diagnosis within 24 hours of QF-PCR and the advantage of evaluating the chromosome structure with the karyotype.
The possibility of replacing direct chorionic biopsy culture with QF-PCR + long chorionic culture was raised.
The molecular techniques of QF-PCR, prenatal karyotype and prenatal microarray are complementary.
It is concluded that in all pregnant patients with an altered combined screening of the first trimester or the presence of fetal malformations, or a positive Fetal DNA Test (NIPT) an invasive diagnostic test (chorionic biopsy or amniocentesis) is indicated.
In every prenatal sample, QF-PCR and prenatal karyotype should be performed. If the QF-PCR identifies an aneuploidy, this result can be reported after 24 hours, while the karyotype is awaited to define whether it is a structural abnormality or a free trisomy.
If the QF-PCR results normal, and an aneuploidy is suspected in another chromosome different from those evaluated by this technique, such anomaly will be identified in the karyotype.
If both studies were normal, or a microdeletion/microduplication syndrome was suspected, the prenatal microarray study would proceed.
It is concluded that this proposal for a combination of screening methods and molecular techniques allows a greater performance of prenatal diagnosis, to define prognosis, counseling and therapeutic conduct in an early and efficient manner. |
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